Bubble manufacturing container

ABSTRACT

A bubble manufacturing container  20  of the present invention includes: a container body  21  having an opening portion; and a rubber stopper  221  provided on the opening portion of the container body  21 . The rubber stopper  221  is constituted so that the bubbles  1  of an inside of the container body  21  are able to be taken by piercing an injection needle. It is preferred that the bubble manufacturing container  20  has a fastening portion  222  provided on the rubber stopper  221 , having an opening and sealing the container body  21  with the rubber stopper  221 . Furthermore, it is preferred that the container body  21  mounts a weight portion.

TECHNICAL FIELD

The present invention relates to a bubble manufacturing container usedfor a method for manufacturing microbubbles or nanobubbles.Particularly, the present invention relates to a bubble manufacturingcontainer used for a method for manufacturing microbubbles ornanobubbles used for ultrasonic diagnosis and ultrasound therapy.

RELATED ART

In recent years, in the various fields such as medical care, food,seafood farming, and waste water treatment, the use of micro-sized(about hundreds of micrometers) or nano-sized (equal to or smaller thanhundreds of nanometers) bubbles has been examined. Particularly, in themedical field, a method is known in which an ultrasonic diagnosis ismade for chest or abdomen by using the microbubbles as an ultrasoundcontrast agent.

The ultrasonic diagnosis method is a method of making a diagnosis byinjecting an ultrasound contrast agent into the body through a vein orthe like, irradiating a diagnosis site with ultrasonic waves, and makingreflected waves (reflection echo) from the ultrasound contrast agentinto an image. As the ultrasound contrast agent, minute air bubbles(microbubbles) each composed of an outer shell constituted of a protein,a lipid, or the like and a gas sealed in the outer shell are widelyused.

In recent years, an ultrasound therapy method using the microbubbles hasbeen examined (for example, PTL 1). More specifically, microbubbles inwhich a gene or a medical agent (drug) is sealed are injected into thebody and transported to an affected site through blood vessels. When themicrobubbles reach the vicinity of the affected site, ultrasonic wavesare radiated to the microbubbles such that the microbubbles burst. Inthis way, the drug sealed in the microbubbles can be intensivelyadministered to the affected site.

As methods for manufacturing the microbubbles, a supersaturation bubblegeneration method and a gas-liquid two-phase flow swirling method areknown. The supersaturation bubble generation method is a method in whicha gas is dissolved under a high pressure in a mixed liquid containingthe constituent materials of the microbubbles and physiological saline,and then the pressure is reduced such that the microbubbles aregenerated in the mixed liquid. The gas-liquid two-phase flow swirlingmethod is a method in which the aforementioned mixed liquid is stirredat a high speed such that the mixed liquid swirls, a gas is allowed tosufficiently drawn into the swirl, and then the swirl is stopped suchthat the microbubbles are generated in the mixed liquid.

In the aforementioned methods for manufacturing microbubbles, in orderto generate the microbubbles, at least 1 to 10 L of the mixed liquidneeds to be prepared. Furthermore, it is difficult to stably generatethe microbubbles by using a small amount of mixed liquid (for example,several milliliters of the mixed liquid). In addition, unfortunately,the generated microbubbles vary in size.

CITATION LIST Patent Literature

[PTL 1] JP-A-2002-209896

SUMMARY OF INVENTION Technical Problem

The present invention has been made in consideration of theaforementioned problems of the related art, and an object thereof is toprovide a bubble manufacturing container that can stably manufacturebubbles (microbubbles or nanobubbles) having a uniform size.

Solution to Problem

The aforementioned objects are achieved by the present inventiondescribed below in (1) to (14).

(1) A bubble manufacturing container used for manufacturing bubbles,comprising:

a container body having an opening portion; and

a rubber stopper provided on the opening portion of the container body,

wherein the rubber stopper is constituted so that the bubbles of aninside of the container body are able to be taken by piercing aninjection needle.

(2) The bubble manufacturing container described in (1) furthercomprising a fastening portion provided on the rubber stopper, having anopening and sealing the container body with the rubber stopper,

wherein the container body mounts a weight portion.

(3) The bubble manufacturing container described in (2),

wherein the container body is constituted from a top body portion havingthe opening portion and a bottom body portion mounting the weightportion, and

wherein the bottom body portion has an inner diameter smaller than aninner diameter of the top body portion.

(4) The bubble manufacturing container described in (3),

wherein the top body portion has a diameter-reduced portion that theinner diameter of the top body portion is reduced so as to become theinner diameter of the bottom body portion.

(5) The bubble manufacturing container described in (3) or (4),

wherein the bottom body portion has a screw groove formed over anentirety of an outer circumferential surface of the bottom body portion,and

wherein the weight portion is constituted to screw with the screw grooveand be movable on the bottom body portion.

(6) The bubble manufacturing container described in (2),

wherein the weight portion is provided in a vicinity of the openingportion of the container body and has a through hole to be pierced bythe injection needle, and the through hole corresponds to the opening ofthe fastening portion.

(7) The bubble manufacturing container described in (6),

wherein the weight portion is provided on the container body to coverthe opening portion, and

wherein the rubber stopper is provided on the weight portion and has amark to be pierced by the injection needle at a position to correspondto the through hole.

(8) The bubble manufacturing container described in (7),

wherein the position of the mark of the rubber stopper is constituted tobe shifted from a position of the through hole of the weight portion byrotating the fastening portion.

(9) The bubble manufacturing container described in (1),

wherein the container body has a long shape, both end portions and aprojection portion formed between the both end portions, and thecontainer body has two weight portions covering the both end portions ofthe container body, and

wherein the opening portion is provided on the projection portion.

(10) The bubble manufacturing container described in (8),

wherein the container body is formed into a cylindrical shape to openthe both end portions to an outside.

(11) The bubble manufacturing container described in any one of (2) to(10),

wherein the weight portion is constituted of a material having a densityhigher than a density of a material constituting the container body.

(12) The bubble manufacturing container described in any one of (1) to(11) further comprising:

a mininert valve for maintaining a sealing property of the inside of thecontainer body; and

a tube for communicating the inside of the container body with themininert valve.

(13) The bubble manufacturing container described in (12),

wherein the mininert valve has a duct communicating with the tube andallowing the injection needle to be pierced and an opening and closingmechanism controlling an opening and closing of the duct, and

wherein the tube connects to the rubber stopper or the container body.

(14) The bubble manufacturing container described in any one of (1) to(13),

wherein the container body has an inner surface, and at least a part ofthe inner surface is in a form of a concave surface, a convex surface ora corrugated surface.

Advantageous Effects of Invention

According to the present invention, by using the bubble manufacturingcontainer, a large amount of bubbles having a uniform size can be stablygenerated in an aqueous liquid. As a result, it is possible to provide acontainer containing the large amount of bubbles having the uniformsize.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view for illustrating an example of bubbles manufactured bya method for manufacturing bubbles of the present invention.

FIG. 2 shows perspective views illustrating a state where a portion ofan example of bubbles manufactured by the method for manufacturingbubbles of the present invention is cut. FIG. 1(a) shows a state where aportion of a bubble in which a gas is sealed in an outer shell is cut,and FIGS. 1(b) and 1(c) each show a state where a portion of a bubble inwhich a gas and a drug are sealed in an outer shell is cut.

FIG. 3 is a flow chart for illustrating a first embodiment of the methodfor manufacturing bubbles of the present invention.

FIG. 4 shows cross-sectional views for illustrating the first embodimentof the method for manufacturing bubbles of the present invention.

FIG. 5 is a partially enlarged view for illustrating a state where anaqueous liquid violently collides with an inner surface (top surface) ofa container in a step of vibrating a container shown in FIG. 4(c).

FIG. 6 is a flow chart for illustrating a second embodiment of themethod for manufacturing bubbles of the present invention.

FIG. 7 shows cross-sectional views for illustrating the secondembodiment of the method for manufacturing bubbles of the presentinvention.

FIG. 8 is a flow chart for illustrating a fifth embodiment of the methodfor manufacturing bubbles of the present invention.

FIG. 9 is a partial cross-sectional view showing the vicinity of a lidof a manufacturing container used in a sixth embodiment of the methodfor manufacturing bubbles of the present invention.

FIG. 10 shows cross-sectional views schematically showing containersused in a seventh embodiment of the method for manufacturing bubbles ofthe present invention.

FIG. 11 shows perspective views for illustrating an eighth embodiment ofthe method for manufacturing bubbles of the present invention.

FIG. 12 shows views for illustrating the constitution (a handle is nowshown) of the vicinity of a rubber stopper of a Mininert valve shown inFIG. 11(a). FIG. 12(a) is a top view of the vicinity of the rubberstopper of the Mininert valve, and FIG. 12(b) is a cross-sectional viewof FIG. 12(a) taken along the line X-X.

FIG. 13 is a cross-sectional view of a bubble-containing container shownin FIG. 11(c).

FIG. 14 is a perspective view for illustrating a manufacturing containerused in a ninth embodiment of the method for manufacturing bubbles ofthe present invention.

FIG. 15 is a cross-sectional view for illustrating a manufacturingcontainer used in a tenth embodiment of the method for manufacturingbubbles of the present invention.

FIG. 16 shows cross-sectional views for illustrating a manufacturingcontainer used in an eleventh embodiment of the method for manufacturingbubbles of the present invention. FIG. 16(a) shows the manufacturingcontainer in a disassembled state, and FIG. 16(b) shows themanufacturing container in an assembled state.

FIG. 17 is a cross-sectional view for illustrating a manufacturingcontainer used in a twelfth embodiment of the method for manufacturingbubbles of the present invention.

FIG. 18 shows cross-sectional views for illustrating a manufacturingcontainer used in a thirteenth embodiment of the method formanufacturing bubbles of the present invention. FIG. 18(a) shows themanufacturing container in a disassembled state, and FIG. 18(b) showsthe manufacturing container in an assembled state.

FIG. 19 shows views for illustrating positions of an opening portionformed in a lid of the manufacturing container shown in FIG. 18(b). FIG.19(a) is a view for illustrating a state where an injection needle of asyringe is not yet pierced into a rubber stopper, and FIG. 19(b) is aview for illustrating a state where a fastening portion is fastened to abottom plate portion after the injection needle is pulled out of therubber stopper.

FIG. 20(a) is a graph showing a bubble diameter distribution of bubblesobtained when the bubbles are manufactured at the number of revolutionof each of 5,000 rpm and 6,500 rpm. FIG. 20(b) is a partially enlargedview obtained by setting the range of the abscissa axis in the graphshown in FIG. 20(a) to be 0 to 700 nm.

FIG. 21(a) is a graph showing a relationship between the number ofrevolution of a sealed vial and an average bubble diameter. FIG. 21(b)is a graph showing the relationship between the number of revolution ofa sealed vial and a content of bubbles.

FIG. 22(a) is a graph showing a relationship between a volume of a gassealed in a sealed vial and an average bubble diameter. FIG. 22(b) is agraph showing a relationship between a volume of a gas sealed in asealed vial and a content of bubbles.

FIG. 23 shows fluorescent micrographs of a culture medium ofcerebrovascular pericytes cultured for 48 hours at 37° C. FIG. 23(a) isan image of a sample irradiated with ultrasonic waves at an irradianceof 0.6 W/cm², and FIG. 23(b) is an image of a sample irradiated withultrasonic waves at an irradiance of 0.8 W/cm².

FIG. 24 shows fluorescent micrographs of a culture medium ofcerebrovascular pericytes cultured for 48 hours at 37° C. FIG. 24(a) isan image of a sample irradiated with ultrasonic waves at an irradianceof 0.9 W/cm², and FIG. 24(b) is an image of a sample irradiated withultrasonic waves at an irradiance of 1.0 W/cm².

FIG. 25 is a graph showing bubble diameter distributions of bubblesobtained in Examples 4 to 11.

FIG. 26 is a graph showing bubble diameter distributions of bubblesobtained in Examples 12 to 17.

FIG. 27 shows micrographs and bubble diameter distribution graphs ofbubbles obtained in Examples 18 and 19.

FIG. 28 shows micrographs and bubble diameter distribution graphs ofbubbles obtained in Example 20.

DESCRIPTION OF EMBODIMENTS

Hereinafter, bubbles, a method for manufacturing bubbles, and a bubblemanufacturing container of the present invention will be described basedon suitable embodiments shown in the attached drawings.

1. Bubbles

First, prior to the description of the method for manufacturing bubblesand a bubble manufacturing container of the present invention, bubblesmanufactured by the method for manufacturing bubbles of the presentinvention (bubbles of the present invention) will be described.

FIG. 1 is a view for illustrating an example of bubbles manufactured bythe method for manufacturing bubbles of the present invention. FIG. 2shows perspective views illustrating a state where a portion of anexample of bubbles manufactured by the method for manufacturing bubblesof the present invention is cut. FIG. 2(a) shows a state where a portionof a bubble in which a gas is sealed in an outer shell is cut, and FIGS.2(b) and 2(c) show a state where a portion of bubbles in which a gas anda drug are sealed in an outer shell is cut.

<First Constitution Example>

First, the bubbles 1 shown in FIG. 1 will be described.

The bubbles 1 (air bubbles) shown in FIG. 1 are formed bymicro-dispersion of a gas 3 in an aqueous liquid 10. The bubbles 1 canbe manufactured by first and second embodiments of the method formanufacturing bubbles of the present invention that will be describedlater. The bubbles 1 can be used in various fields such as medical care,food, seafood farming, and waste water treatment. In the presentembodiment, a case where the bubbles 1 are used as an ultrasoundcontrast agent for ultrasonic diagnosis will be described.

The bubbles 1 constituted as above are formed using an aqueous medium asthe aqueous liquid 10. Examples of the aqueous medium include water suchas distilled water, pure water, ultrapure water, deionized water, and ROwater, physiological saline (saline with a concentration of about 0.9%)such as Saline and phosphate buffered saline (PBS), an aqueous sugarsolution obtained by mixing various sugars such as glucose and sucrosewith distilled water, and the like. One kind of these can be usedsingly, or two or more kinds thereof can be used in combination.

The gas 3 is a substance that is in a gaseous state at the temperature(about 20° C.) at the time of manufacturing the bubbles 1. Furthermore,the gas 3 is the substance that is in the gaseous state even in a statewhere the bubbles 1 are injected into the body, that is, even at thebody temperature (about 37° C.).

The gas 3 is not particularly limited, and examples thereof include: aninert gas such as air, nitrogen, nitrous oxide, oxygen, carbon dioxide,hydrogen, helium, argon, xenon, and krypton; sulfur fluoride such assulfur hexafluoride, disulfur decafluoride, and trifluoromethyl sulfurpentafluoride; low-molecular weight hydrocarbons and halides thereofsuch as methane, ethane, propane, butane, pentane, cyclopropane,cyclobutane, cyclopentane, ethylene, propylene, propadiene, butene,acetylene, propyne, perfluoropropane, perfluorobutane, andperfluoropentane; ethers such as dimethyl ether; ketones; esters; andthe like. Among these, one kind of substance can be used singly, or twoor more kinds of substances can be used in combination. Among thesesubstances, sulfur hexafluoride, perfluoropropane, perfluorobutane, andperfluoropentane are particularly preferable. The bubbles 1 in whichthese gases are sealed exhibit high stability in the body and are morereliably transported to an affected site (target site of treatment) or atarget site of diagnosis through blood vessels.

The diameter of the bubbles 1 constituted with the aforementionedcomponents changes with the change of conditions of each step of themethod for manufacturing bubbles of the present invention. That is, thebubbles 1 to be manufactured have microsize (about hundreds ofmicrometers) or nanosize (about hundreds of nanometers).

The average diameter of the bubbles 1 is not particularly limited.However, specifically, the average diameter of the bubbles 1 ispreferably about 10 nm to 1,000 μm, more preferably about 10 nm to 100μm, and even more preferably about 50 nm to 2,000 nm. In a case wherethe average diameter of the bubbles 1 is within the aforementionedrange, because the diameter of the bubbles 1 is small enough, thebubbles 1 can move smoothly in blood vessels due to the blood flow whenbeing injected into the body by intravenous injection. Furthermore, thebubbles having such a diameter exhibit high stability in the bloodvessels and are reliably transported to a target site without beingdestroyed while moving in blood vessels. Particularly, becausenanobubbles exhibit high stability in the blood vessels, they arereliably transported to the target site substantially without beingdestroyed.

Generally, gas-containing bubbles have a property of efficientlyreflecting ultrasonic waves from the interface between a liquid and agas. Therefore, in the bubbles 1 having the average diameter within theaforementioned range, the area of the interface between the liquid(aqueous liquid 10 or blood in a case where the bubbles 1 are injectedinto the body as an ultrasound contrast agent) and the gas 3 is largeenough, and hence the bubbles 1 are effectively used as the ultrasoundcontrast agent.

The bubbles 1 constituted as above can also be used in the fields otherthan a medical field, such as food, seafood farming, and waste watertreatment. Particularly, the stability of the bubbles 1 having theaverage diameter within the aforementioned range can be sufficientlyimproved, and hence it is easy to handle the bubbles 1. Therefore, thebubbles 1 can be used in various fields.

<Second Constitution Example>

Next, the bubble 1 shown in FIG. 2(a) will be described.

Herein, the differences between the bubbles 1 of the first constitutionexample and the bubbles 1 of the second constitution example will bemainly described, and the same details will not be described.

The bubble 1 (air bubble) shown in FIG. 2(a) can be manufactured bythird and fifth to thirteenth embodiments of the method formanufacturing bubbles of the present invention that will be describedlater. The bubble 1 shown in FIG. 2(a) has an outer shell 2 (sphericalmembrane) constituting a shell of the bubble 1 and the gas 3 sealed inthe outer shell 2. Such a bubble 1 can be used in various fields such asmedical care, food, seafood farming, and waste water treatment. In thepresent embodiment, a case where the bubbles 1 are used as an ultrasoundcontrast agent for ultrasonic diagnosis will be described. Hereinafter,each component constituting the bubbles 1 will be described.

The outer shell 2 functions to retain the gas 3 sealed therein withinthe bubble 1.

The outer shell 2 is mainly constituted with an amphipathic material(outer shell material) having properties (substituents) of showing boththe hydrophobicity and the hydrophilicity in a single molecule. Theamphipathic material is not particularly limited, and examples thereofinclude: a protein such as albumin, a phospholipid such as apolycationic lipid, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, and phosphatidalethanolamine; a higher fattyacid such as palmitic acid and stearic acid; sugars such as galactose;sterols such as cholesterol and sitosterol; a surfactant; a natural orsynthetic polymer; a fluorescent dye; an antibody; a labeling metal; andthe like. Among these, one kind of material can be used singly, or twoor more kinds of materials can be used in combination.

The amphipathic material constituting the outer shell 2 is disposed inthe form of a sphere in an aqueous medium such that a hydrophobic groupbecomes inside and a hydrophilic group becomes outside, although such aproperty is not shown in FIG. 2. Due to this property, the outer shell 2becomes a micelle constituted with a monolayer of a molecule of theamphipathic material or becomes a liposome (spherical molecularmembrane) constituted with a bilayer of the molecule of the amphipathicmaterial.

The diameter of the bubble 1 constituted with the aforementionedcomponents is the same as the diameter of the bubble 1 shown in FIG. 1.

Generally, a bubble containing a gas in an outer shell has a property ofefficiently reflecting ultrasonic waves from the interface between theouter shell and the gas. Therefore, in the bubbles 1 having the averagediameter with the aforementioned range, the area of the interfacebetween the outer shell 2 and the gas 3 is large enough, and hence thebubbles 1 are effectively used as the ultrasound contrast agent.

<Third Constitution Example>

Next, the bubbles 1 shown in FIGS. 2(b) and 2(c) will be described.

Herein, the differences between the bubbles of the first and secondconstitution examples and the bubbles of the present embodiment will bemainly described, and the same details will not be described.

The bubbles 1 shown in FIGS. 2(b) and 2(c) can be manufactured by fourthto thirteenth embodiments of the method for manufacturing bubbles of thepresent invention that will be described later. Such a bubble 1 has theouter shell 2 constituting a shell of the bubble 1 and a gas 3 and adrug 4 sealed in the outer shell 2. The bubble 1 is used for ultrasoundtherapy and ultrasonic diagnosis. FIG. 2(b) shows the bubble 1 in whichthe drug 4 is sealed in the outer shell 2 in a gaseous state or a solidstate, and FIG. 2(c) shows the bubble 1 in which the drug 4 is sealed inthe outer shell 2 in a liquid state.

The outer shell 2 functions to retain the gas 3 or the drug 4 sealedtherein within the bubble 1 and to protect the drug 4 until the bubble 1is transported to an affected site.

In the bubbles 1 shown in FIGS. 2(b) and 2(c), the drug 4 is an activecomponent for treating various diseases such as prostate cancer, uterinemyoma, myocardial infarction, and cerebral infraction. The drug 4 istransported to the affected site in a state of being contained in thebubble 1, and the outer shell 2 bursts in the vicinity of the affectedsite by being irradiated with ultrasonic waves. In this way, the drug 4is administered to the affected site. The drug 4 may be contained in theouter shell 2 itself or adsorbed onto an outer surface of the outershell 2, although this constitution is not shown in the drawings.

The drug 4 is not particularly limited as long as it is effective fortreating the diseases, and includes a gene, a medical agent, and thelike. Specific examples thereof include a peptide, an antibody,oligosaccharide, polysaccharide, a gene, oligonucleotide, antisenseoligonucleotide, siRNA, ribozyme, a triple helix molecule, a viralvector, a plasmid, a low-molecular weight organic compound, ananticancer drug, a metal, and the like. Among these, one kind of drugcan be used singly, or two or more kinds of drugs can be used incombination.

The volume ratio between the drug 4 and the gas 3 is preferably about1:99 to 90:10, more preferably about 10:90 to 70:30, and even morepreferably about 40:60 to 60:40. In a case where the volume ratiobetween the drug 4 and the gas 3 is within the aforementioned range, thestability of the bubble 1 can be improved, and hence the bubble 1 can bemore reliably transported to the vicinity of the affected site.Furthermore, when the outer shell 2 bursts in the vicinity of theaffected site, a sufficient amount of drug can be administered to theaffected site. Therefore, the affected site can be more efficientlytreated.

Similarly to the bubble 1 shown in FIG. 2(a), the diameter of the bubble1 constituted with the aforementioned components changes with the changeof the conditions of each step of the method for manufacturing bubblesof the present invention.

At an affected site where a cancer cell exists, neovessels having adiameter smaller than that of a normal vessel extend to the cancer cellfrom the peripheral blood vessels of the affected site. In a case wherethe bubbles 1 have an average diameter of about 200 to 300 nm, thebubbles 1 can be smoothly transported even in the neovessels and canreach the cancer cell. That is, those bubbles 1 can be suitably used forcancer treatment. Furthermore, it is possible to cause some of thebubbles 1 to pass through the vessel wall and to be incorporated intothe cancer cell.

In a case where the bubbles 1 have an average diameter of about 600 to900 nm, the bubbles 1 can be smoothly transported in blood vessels inthe brain, and the position thereof can be clearly specified in anultrasonic image. Therefore, the bubbles 1 can be suitably used in braintreatment (for example, endovascular treatment of brain).

The average diameter of the bubbles 1 shown in FIG. 1 and FIGS. 2(a) to2(c) can be measured by observation using, for example, a laserdiffraction-scattering method, a nanoparticle tracking analysis method,an electric resistance method, an atomic force microscope (AFM), a lasermicroscope, and the like. As a device for measuring by AFM, for example,it is possible to use a resonant particle measurement system (tradename: ARCHIMEDES) manufactured by Malvern Instruments Ltd.

The bubbles 1 described above can be manufactured by the method formanufacturing bubbles of the present invention that will be describedbelow. Hereinafter, the method for manufacturing bubbles of the presentinvention will be specifically described.

2. Method for Manufacturing Bubbles

First Embodiment

Next, a first embodiment of the method for manufacturing bubbles of thepresent invention will be described. The bubbles 1 shown in FIG. 1described above can be manufactured by the method for manufacturingbubbles of the present embodiment.

FIG. 3 is a flow chart for illustrating the first embodiment of themethod for manufacturing bubbles of the present invention. FIGS. 4(a) to4(d) are cross-sectional views of a manufacturing container forillustrating the first embodiment of the method for manufacturingbubbles of the present invention. FIG. 5 is a partially enlarged viewfor illustrating a state where an aqueous liquid violently collides withan inner surface (top surface) of a container in a step of vibrating acontainer shown in FIG. 4(c).

In the following description, the upper side in each of FIGS. 4(a) to4(d) and FIG. 5 will be referred to as “top”, and the lower side in eachof FIGS. 4(a) to 4(d) and FIG. 5 will be referred to as “bottom”.

As shown in FIG. 3, the method for manufacturing bubbles of the presentembodiment includes five steps consisting of Steps (S1) to (S5). Step(S1) is a step of preparing an aqueous liquid and a bubble manufacturingcontainer (hereinafter, simply referred to as “manufacturing container”)into which the aqueous liquid is injected. Step (S2) is a step ofinjecting the aqueous liquid into the manufacturing container to apredetermined height. Step (S3) is a step of sealing the manufacturingcontainer in a state where the manufacturing container is filled with agas. Step (S4) is a step of vibrating the manufacturing container at apredetermined number of revolution such that the aqueous liquidrepeatedly collides with the inner surface of the container. Step (S5)is a step of allowing the manufacturing container to stand. Hereinafter,these steps will be sequentially described.

[S1] Preparation Step

First, the aqueous liquid 10 is prepared.

In the method for manufacturing bubbles of the present embodiment, asthe aqueous liquid 10, the aqueous medium described above is used.

The inventor of the present invention found that the higher theconcentration water in the aqueous liquid 10 becomes, the smaller thediameter of the bubbles 1 generated becomes, and the greater the amountof the bubbles 1 generated becomes. Accordingly, in a case where water(distilled water) is used as the aqueous liquid 10, it is possible togenerate more bubbles 1 having a smaller diameter.

In a case where an aqueous sugar solution is used as the aqueous liquid10, the lower the concentration of sugar in the aqueous sugar solutionbecomes, that is, the higher the concentration of water becomes, thesmaller the diameter of the bubbles 1 generated becomes, and the greaterthe amount of the bubbles 1 generated becomes. Accordingly, byappropriately setting the type of the aqueous medium and the conditionof Step (S4), it is possible to obtain the bubbles 1 having an intendeddiameter.

The concentration of sugar in the aforementioned aqueous sugar solutionis not particularly limited, but is preferably about 0.01 to 60 wt %,more preferably about 0.1 to 50 wt %, and even more preferably about 5to 30 wt %. In a case where the aqueous sugar solution in which theconcentration of sugar is within the aforementioned range is used, inStep (S4) which will be described later, the stability of the bubbles 1generated in the aqueous liquid 10 is improved. Therefore, the bubbles 1are more reliably prevented from accidentally bursting, and temporalstability of the bubbles 1 is improved.

Then, a manufacturing container 20 (first embodiment of the bubblemanufacturing container) is prepared.

The manufacturing container 20 includes a container body 21accommodating the aqueous liquid 10 and having an opening portion, and alid 22 for sealing the container body 21.

The container body 21 is not particularly limited, but preferably lookslike a bottomed cylinder as shown in FIG. 4(a). In the presentembodiment, as the container body 21, a vial having a volume of about0.5 to 20 ml is used. In the method for manufacturing bubbles of thepresent invention, even in a case where such a vial with a small volumeis used as the container body 21, when the container body 21 is sealedwith the lid 22, an appropriate pressure is applied to the aqueousliquid 10 within the sealed space in the container body 21. Accordingly,the bubbles 1 having a uniform size can be stably obtained.Particularly, in a case where a vial having a volume of about 0.5 to 1.5ml is used, in a single manufacturing container 20, a bubble-containingliquid of about 0.3 to 0.6 ml that is a volume necessary for a singlesession of ultrasonic diagnosis can be manufactured. In this case, atthe time of ultrasonic diagnosis, the bubble-containing liquid in asingle manufacturing container 20 can be used up. Therefore, it ispossible to eliminate a waste of the manufactured bubble-containingliquid.

The vial having such a small volume (volume: about 0.5 to 20 ml) hasdimensions in which a length X in a longitudinal direction is about 35to 60 mm and an outer diameter R is about 10 to 40 mm.

As shown in FIGS. 4(b) to 4(d), the lid 22 includes a disk-like rubberstopper (septum) 221 that adheres to a vial mouth of the container body21 and a fastening portion 222 that fixes the rubber stopper 221 to thevial mouth of the container body 21.

The rubber stopper 221 is not particularly limited, but for example, arubber stopper made of silicon can be used.

The fastening portion 222 is constituted such that it covers the edge ofthe rubber stopper 221. When seen in a plan view, the fastening portion222 has an opening approximately at the center thereof. On the innercircumferential surface of the fastening portion 222 on the vial mouthside and on the outer circumferential surface of the container body 21on the vial mouth side, screw grooves that can be screwed with eachother are formed (not shown in the drawing). By screwing the screwgrooves with each other, the rubber stopper 221 is fixed to the vialmouth of the container body 21 in a state of adhering to the vial mouth.Furthermore, by caulking the vial mouth of the container body 21 withthe fastening portion 222, the container body 21 and the fasteningportion 222 can be fixed to each other in a state where the rubberstopper 221 adheres to the vial mouth of the container body 21.

[S2] Step of Injecting Aqueous Liquid into Manufacturing Container

The prepared aqueous liquid 10 is injected into the container body 21(manufacturing container 20) to a predetermined height. In the presentembodiment, as shown in FIG. 4(a), the liquid is injected to Y [mm].Accordingly, as shown in FIG. 4(a), the container body 21 into which theaqueous liquid 10 is injected has a void portion 11 on the top portionthereof.

In the present embodiment, in a state where the container body 21(manufacturing container 20) into which the aqueous liquid 10 isinjected is allowed to stand horizontally, provided that the height(length in the longitudinal direction) of the container body 21 is X[mm] and the level of the surface of the aqueous liquid 10 in thecontainer body 21 is Y [mm], it is preferable that a relationship of0.2≤Y/X≤0.7 is satisfied. In a case where the aforementionedrelationship is satisfied, due to the existence of the void portion 11that is large enough, in Step (S4), it is possible to make the aqueousliquid 10 more violently collide with the top and bottom surfaces andthe lateral surface (particularly, the top and bottom surfaces) of themanufacturing container 20. Due to the collision, shock waves occur inthe aqueous liquid 10, and hence the bubbles 1 can be easily formed inthe aqueous liquid 10.

The aforementioned X and Y more preferably satisfy a relationship of0.3≤Y/X≤0.5, and even more preferably satisfy a relationship of0.35≤Y/X≤0.4. In this way, in Step (S4), bubbles can be more easilyformed in the aqueous liquid 10.

[S3] Step of Sealing Manufacturing Container

Then, the container body 21 is sealed in a state of being filled withthe gas 3 (see FIG. 4(b)). Specifically, in the void portion 11 of thecontainer body 21 into which the aqueous liquid 10 is injected, purgingis performed using the gas 3, and then the lid 22 is fastened to theopening portion (vial mouth) of the container body 21. In this way, theaqueous liquid 10 and the gas 3 are sealed in the manufacturingcontainer 20.

As a method for performing the purging in the void portion 11 of thecontainer body 21 by using the gas 3, for example, the container body 21into which the aqueous liquid 10 is injected is moved into a chamber.Thereafter, the air in the chamber is substituted with the gas 3, andthen the lid 22 is fastened to the opening portion of the container body21. In this way, the aqueous liquid 10 and the gas 3 can be sealed inthe manufacturing container 20.

As the gas 3, the various gases described above are used.

[S4] Step of Vibrating Manufacturing Container

Then, the manufacturing container 20 is vibrated such that the aqueousliquid 10 repeatedly collides with the top and bottom surfaces and thelateral surface (particularly, the top and bottom surfaces) of themanufacturing container 20. In the present embodiment, as shown in FIG.4(c), the manufacturing container 20 is vibrated such that the containerreciprocates approximately in the longitudinal direction (a verticaldirection in FIG. 4(c)) thereof.

In this step, the manufacturing container 20 (lower view in FIG. 4(c))sealed in Step (S3) is vibrated upwardly (middle view in FIG. 4(c)). Asa result, the aqueous liquid 10 moves to the vicinity of the middle ofthe manufacturing container 20. In a case where the manufacturingcontainer 20 is further vibrated upwardly, the aqueous liquid 10 movesto the top portion of the manufacturing container 20 and collides withthe bottom surface (rubber stopper 221) of the lid 22 (upper view inFIG. 4(c)). At this time, as shown in FIG. 5, shock waves occur. Due tothe pressure of the shock waves, the gas 3 is micro-dispersed in theaqueous liquid 10, and hence the bubbles 1 are formed. The bubbles 1contain the gas 3 in which the aqueous liquid 10 is micro-dispersed ordissolved due to vibration.

Meanwhile, the manufacturing container 20 (upper view in FIG. 4(c)) isvibrated downwardly (middle view in FIG. 4(c)). As a result, the aqueousliquid 10 moves to the vicinity of the middle of the manufacturingcontainer 20. In a case where the manufacturing container 20 is furthervibrated downwardly, the aqueous liquid 10 moves to the bottom portionof the manufacturing container 20 and collides with the bottom surfaceof the manufacturing container 20 (lower view in FIG. 4(c)). At thistime, as shown in FIG. 5, the shock waves also occur.

When the manufacturing container 20 is vibrated in the verticaldirection, the aqueous liquid 10 also collides with the inner lateralsurface of the manufacturing container 20. At this time, as shown inFIG. 5, the shock waves also occur.

By repeatedly performing the aforementioned operation, it is possible tostably generate a large amount of bubbles 1 having a uniform size in theaqueous liquid 10.

In the method for manufacturing bubbles of the present invention, inorder to obtain the bubbles 1 that are fine enough and have a uniformdiameter, the manufacturing container 20 is vibrated at the number ofrevolution of equal to or higher than 5,000 rpm. As a result, themagnitude (pressure) of the shock waves that occur when the aqueousliquid 10 collides with the manufacturing container 20 becomes highenough, fine bubbles 1 are generated in the aqueous liquid 10, and thediameter of the bubbles can be made uniform. In a case where the numberof revolution of the manufacturing container 20 is set to be low withinthe aforementioned range, the magnitude of the occurring shock waves isreduced, and hence the bubbles 1 having a relatively large diameter canbe generated. Furthermore, in a case where the number of revolution isset to be high, the magnitude of the occurring shock waves increases,and hence the bubbles 1 having a relatively small diameter can begenerated. In the present specification, “number of revolution” of themanufacturing container 20 refers to the number of times that themanufacturing container 20 travels the whole vibration route thereof perunit time. For example, in a case where the manufacturing container 20vibrates at 5,000 rpm, it means that the manufacturing container 20travels (vibrates) in 5,000 times the whole vibration route for 1minute.

The number of revolution of the manufacturing container 20 is morepreferably equal to or higher than 5,500 rpm, and even more preferably6,000 to 20,000 rpm. In a case where the number of revolution of themanufacturing container 20 is within the aforementioned range, it ispossible to more reliably prevent the bubbles 1 generated by vibrationfrom being destroyed due to collision or from coarsening by beingcombined with each other. As a result, it is possible to generate alarge amount of bubbles 1 having a more uniform diameter in the aqueousliquid 10 with reducing the diameter of the bubbles 1.

As a device that can vibrate the manufacturing container 20 at thenumber of revolution described above, for example, a bead-typehigh-speed cell disruption system (homogenizer) can be used.Specifically, Precellys manufactured by bertin Technologies and the likecan be used.

The pressure of the shock waves that occur when the aqueous liquid 10collides with the manufacturing container 20 is preferably 40 kPa to 1GPa. In a case where the pressure of the shock waves that occur at thetime of collision between the aqueous liquid 10 and the manufacturingcontainer 20 is within the aforementioned range, the bubbles 1 generatedin the aqueous liquid 10 become finer, and the size thereof can be mademore uniform. Particularly, the higher the pressure of the shock wavesthat occur at the time of collision between the aqueous liquid 10 andthe manufacturing container 20 is, the finer the generated bubbles 1 canbecome.

At the time of vibrating the manufacturing container 20, a vibrationwidth of the manufacturing container 20 in the longitudinal direction ispreferably about 0.7X to 1.5X [mm], and more preferably about 0.8X to 1X[mm]. In this way, when the manufacturing container 20 vibrates, it ispossible to cause the aqueous liquid 10 to reliably collide with thebottom surface of the manufacturing container 20 and the lid 22, and tosufficiently increase the number of times the aqueous liquid 10 collideswith the bottom surface of the manufacturing container 20 and the lid22. Furthermore, in a case where the manufacturing container 20 isvibrated at the sufficient vibration width described above, the speed atwhich the aqueous liquid 10 moves in the manufacturing container 20increases. Therefore, the magnitude of the shock waves that occur whenthe aqueous liquid 10 collides with the bottom surface of themanufacturing container 20 and the lid 22 sufficiently increases. As aresult, it is possible to generate a large amount of fine bubbles 1 inthe aqueous liquid 10.

When the manufacturing container 20 is reciprocated in the verticaldirection, the manufacturing container 20 is preferably vibrated in atransverse direction (horizontal direction) thereof as well. In thisway, the aqueous liquid 10 also collides with the inner lateral surfaceof the manufacturing container 20, and hence more shock waves can begenerated in the aqueous liquid 10. The vibration width of themanufacturing container 20 in the transverse direction is preferablyabout 0.3X to 0.8X [mm], and more preferably about 0.5X to 0.7X [mm]. Inthis way, the aforementioned effects are more markedly exhibited.

The manufacturing container 20 may be vibrated only in the transversedirection thereof. In this case, the vibration width of themanufacturing container 20 in the transverse direction (horizontaldirection) is preferably the same as the vibration width in theaforementioned transverse direction. In a case where the vibration widthis the same, the aqueous liquid 10 reliably collides with the innerlateral surface of the manufacturing container 20, and hence more shockwaves can occur in the aqueous liquid 10. As a result, a large amount offine bubbles 1 can be generated in the aqueous liquid 10.

In this step, it is preferable to vibrate the manufacturing container 20such that an instantaneous relative speed between the manufacturingcontainer 20 and the aqueous liquid 10 in the manufacturing container 20becomes equal to or higher than 40 km/h when the aqueous liquid 10collides with the top and bottom surfaces and the lateral surface of themanufacturing container 20. It is more preferable to vibrate themanufacturing container 20 such that the instantaneous relative speedbecomes equal to or higher than 50 km/h. In a case where theaforementioned condition is satisfied, it is possible to sufficientlyincrease the pressure of the shock waves that occur when the aqueousliquid 10 collides with the manufacturing container 20. As a result, thebubbles 1 generated in the aqueous liquid 10 become finer, and the sizethereof can be made more uniform.

The period of time for which the manufacturing container 20 is vibratedunder the aforementioned condition is preferably about 10 to 120seconds, and more preferably about 30 to 60 seconds. In a case where thevibration time of the manufacturing container 20 is within theaforementioned range, the number of times that the aqueous liquid 10collides with the manufacturing container 20 sufficiently increases, andhence a large amount of bubbles 1 can be generated in the aqueous liquid10. In a case where the vibration time of the manufacturing container 20is set to be long within the aforementioned range, the amount of bubbles1 generated in the aqueous liquid 10 can be further increased.

The average diameter of the bubbles 1 generated in the aqueous liquid 10can be adjusted by changing the number of revolution of themanufacturing container 20 within the aforementioned range. In thepresent embodiment, by using the aforementioned aqueous medium as theaqueous liquid 10, nanobubbles having a size of about tens of nanometersto hundreds of nanometers can be stably generated.

In the present embodiment, the manufacturing container 20 is vibratedsuch that the container reciprocates practically in the longitudinaldirection thereof, but the method for vibrating the manufacturingcontainer 20 is not limited thereto. For example, the manufacturingcontainer 20 may be vibrated such that the container rotates mainly inthe transverse direction and/or the longitudinal direction thereof. Evenin this case, the aqueous liquid 10 in the manufacturing container 20repeatedly collides with the top and bottom surfaces and the lateralsurface of the manufacturing container 20, and as a result, the shockwaves occur. By using the aforementioned vibrating method, a largeamount of bubbles 1 having a uniform size can also be stably generatedin the aqueous liquid 10.

[S5] Step of Allowing Manufacturing Container to Stand

After the manufacturing container 20 is vibrated under theaforementioned conditions, the manufacturing container 20 is allowed tostand (see FIG. 4(d)). In this way, the large amount of bubbles 1 havingthe uniform size (see FIG. 1) can be stably manufactured in themanufacturing container 20. In addition, the manufacturing container 20containing the large amount of the bubbles 1 having the uniform size isobtained.

It is preferable to perform the aforementioned Step (S2), Step (S3), andStep (S4) by making the temperature of the aqueous liquid 10 remainconstant. In this way, the characteristics (viscosity and the like) ofthe aqueous liquid 10 are stabilized in the manufacturing process of thebubbles, and hence the bubbles 1 having the uniform diameter can bestably generated in the aqueous liquid 10. Examples of the method formaking the temperature of the aqueous liquid 10 remain constant includea method of performing each of the aforementioned Steps (S2) to (S4) ina glove box or a thermostatic bath. Particularly, in the presentembodiment, the manufacturing container 20 is vibrated at a high speedin Step (S4). Therefore, due to the collision between the aqueous liquid10 and the inner surface of the manufacturing container 20, themanufacturing container 20 is easily heated. However, by vibrating themanufacturing container 20 in the thermostatic bath, it is possible toreliably prevent the temperature increase of the aqueous liquid 10. As aresult, the bubbles 1 having the uniform diameter can be stablygenerated in the aqueous liquid 10.

Through the aforementioned Steps (S1) to (S5), the bubbles 1 having anaverage diameter of about 10 nm to 1,000 μm are manufactured. In thepresent embodiment in which the aqueous medium described above is usedas the aqueous liquid 10, it is easy to generate the bubbles 1 having asmall average diameter. Particularly, in the present embodiment, a largeamount of bubbles 1 having an average diameter of 10 nm to 1,000 nm canbe generated.

In the method for manufacturing bubbles of the related art, alarge-scale reflux device or various systems (a tube, a nozzle, acompressor, and the like) constituting a bubble manufacturing device arerequired. Therefore, in a case where bubbles used in the field of foodor medical care are manufactured, it is difficult to maintain a cleanand sterile environment. In contrast, in the present invention, becausethe manufacturing container 20 having high airtightness is used formanufacturing the bubbles 1, in a state where the manufacturingcontainer 20 contains the aqueous liquid 10 and the gas 3, asterilization treatment such as γ-ray sterilization may be performed onthe manufacturing container 20. In this way, the interior of themanufacturing container 20 is sterilized, and hence the bubbles 1 can bemanufactured in a sterile environment. Accordingly, the bubbles 1manufactured in this way can be suitably used in the field of food ormedical care.

The bubbles 1 obtained as above can stably exist in the aqueous liquid10. Therefore, the manufacturing container 20 containing the obtainedbubbles (hereinafter, simply referred to as “bubble-containingcontainer”) can be stored for a long period of time at room temperature.Specifically, the container can be stored for 6 to 24 months.Furthermore, even after the container is stored for such a long periodof time, the stability of the bubbles 1 in the aqueous liquid 10 isstill high. Consequently, the bubble-containing container does not needto be vibrated again and can be directly used. In addition, because themanufacturing container 20 having a small volume is used as amanufacturing container, the unit cost of the bubble-containingcontainer can be reduced. As a result, the bubble-containing containerobtained as above has an advantageous of being easily handled in medicalfacilities and the like.

[S6] Step of Centrifugation Treatment

In the method for manufacturing bubbles of the present embodiment, afterStep (S5), a centrifugation treatment may be performed on thebubble-containing container. By this treatment, the bubbles 1 generatedin the manufacturing container 20 can be separated based on the intendedsize.

Specifically, in a case where the centrifugation treatment is performedon the bubble-containing container, the bubbles 1 having a largediameter tend to move to a top layer of the manufacturing container 20while the bubbles 1 having a small diameter tend to move to a bottomlayer of the manufacturing container 20. Accordingly, in a case wherethe liquid (supernatant) of the top layer of the manufacturing container20 is removed using aspiration means (a syringe, a pipette, or thelike), the average diameter of the bubbles 1 in the bubble-containingliquid remaining in the manufacturing container 20 becomes smaller thanthe average diameter of the bubbles 1 in the bubble-containing liquidobtained after Step (S5). Furthermore, the average diameter of thebubbles 1 in the bubble-containing liquid (supernatant) aspirated by theaspiration means becomes larger than the average diameter of the bubbles1 in the bubble-containing liquid obtained after Step (S5). In this way,by using the centrifugation treatment, the bubbles 1 having a moremonodispersed bubble diameter distribution can be obtained.

Furthermore, by adding a substance, having a specific gravity differentfrom that of the aqueous liquid 10, to the bubble-containing liquid andperforming the centrifugation treatment, the bubbles 1 having the largediameter easily move to the top layer while the bubbles 1 having thesmall diameter easily move to the bottom layer. As a result, the bubbles1 having a more monodispersed bubble diameter distribution can beobtained.

For example, in a case where a bubble-containing liquid containing thebubbles 1 having an average diameter of 600 nm is obtained through Steps(S1) to (S5), by appropriately setting the condition of thecentrifugation treatment, it is possible to obtain a bubble-containingliquid containing the bubbles 1 having an average diameter of 200 to 300nm. In a case where such a bubble-containing liquid is used as theultrasound contrast agent, because there are no bubbles 1 having arelatively large diameter, it is possible to obtain a betterhigh-definition image having a high resolution.

The condition of the centrifugation treatment is appropriately setaccording to the average diameter of the bubbles 1 to be separated. Forexample, the condition is set such that a centrifugal acceleration ofabout 1×g to 22,000×g is applied to the bubble-containing liquid forabout 30 seconds to 24 hours. In a case where the centrifugalacceleration is set to be low (about 1×g to 100×g), by performing thetreatment for a long period of time (for about 12 hours to 24 hours),the bubbles 1 having the more monodispersed bubbles diameterdistribution can be obtained. In a case where the centrifugalacceleration is set to be high (100×g to 22,000×g), by performing thetreatment for a relatively short period of time (for about 30 seconds to12 hours), the bubbles 1 having the more monodispersed bubbles diameterdistribution can be obtained. By performing the centrifugation treatmentunder the aforementioned condition, the bubbles 1 having an intendedaverage diameter can be efficiently separated.

A centrifuge that can perform the centrifugation treatment on thebubble-containing container at the aforementioned centrifugalacceleration is not particularly limited, and for example, a high-speedrefrigerated microcentrifuge such as “TOMY MX-301” (trade name,manufactured by TOMY SEIKO CO., LTD.) can be used. In a case where thehigh-speed refrigerated microcentrifuge is used, by setting the numberof revolution thereof to be about 50 to 2,000 rpm, the centrifugalacceleration (centrifugal force) within the aforementioned range isapplied to the bubble-containing liquid.

The centrifugation treatment may be performed once or plural times.

3. How to Use

The bubble-containing container obtained as above is used for making anultrasonic diagnosis for patients.

Specifically, first, an injection needle of a syringe is pierced intothe rubber stopper 221 of the lid 22. Then, the bubble-containing liquidis aspirated from the interior of the bubble-containing container.Thereafter, the injection needle is pulled out of the rubber stopper221, and a blood vessel (for example, a vein) of a patient is piercedwith the injection needle of the syringe into which thebubble-containing liquid is aspirated, such that the bubble-containingliquid is injected into the blood vessel. In this way, the bubbles 1 aretransported to an affected site through the blood flow. The lid 22 maybe removed from the bubble-containing container (manufacturing container20), and the bubble-containing liquid may be aspirated from the interiorof the bubble-containing container by using a syringe.

During the ultrasonic diagnosis, at the timing when the bubbles 1 reacha target site of diagnosis, the bubbles 1 are irradiated with ultrasonicwaves for diagnosis having a frequency and an intensity at which thebubbles 1 may not burst (the ultrasonic waves are radiated to thebubbles 1). Then, the signals (reflection echo) reflected from thetarget site of diagnosis are received and subjected to data processing,thereby imaging the target site of diagnosis. In this way, an ultrasonicdiagnosis can be made.

As a device performing the irradiation of the ultrasonic waves andreceiving the reflection waves from the bubbles 1, known ultrasonicprobes can be used.

The bubble-containing container obtained as above can be used in variousfields, in addition to be used for the ultrasonic diagnosis. Forexample, the bubbles 1 in the bubble-containing container obtained asabove exhibit a germicidal effect with respect to water or food and havean effect of keeping the freshness of food. Furthermore, in a liquidcontaining the bubbles 1, water, and oil (hydrophobic component), alarge amount of oil can be mixed with water. By exploiting such aneffect, it is possible to cook with inhibiting the separation of waterfrom oil in food. Accordingly, the obtained bubble-containing liquid canalso be used in the field of food.

In the above description, by performing Steps (S1) to (S5), the largeamount of the bubbles 1 (see FIG. 1) having the uniform size can bestably manufactured in the manufacturing container 20. However, themethod for manufacturing bubbles of the present embodiment is notlimited thereto. For example, after Step (S5), Step (S4) and Step (S5)may be repeated at least once or more times. By repeating Step (S4) andStep (S5), the bubbles 1 having the uniform diameter can be more stablygenerated.

Second Embodiment

Next, a second embodiment of the method for manufacturing bubbles andthe bubble manufacturing container of the present invention will bedescribed.

FIG. 6 is a flow chart for illustrating the second embodiment of themethod for manufacturing bubbles of the present invention. FIGS. 7(a) to7(d) are cross-sectional views for illustrating the second embodiment ofthe method for manufacturing bubbles of the present invention.

In the following description, the upper side in each of FIGS. 7(a) to7(d) will be referred to as “top”, and the lower side in each of FIGS.7(a) to 7(d) will be referred to as “bottom”.

Hereinafter, regarding the method for manufacturing bubbles of thesecond embodiment, the differences between the method for manufacturingbubbles of the first embodiment and the method for manufacturing bubblesof the present embodiment will be mainly described, and the same detailswill not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the method for manufacturing bubbles of the first embodimentdescribed above, except that in Step (S3) of the first embodiment, themanufacturing container is sealed in a state where the interior of themanufacturing container is pressurized as shown in FIG. 6.

[S3] Step of Sealing Manufacturing Container

In a state where the container body 21 is filled with the gas 3 suchthat the interior of the manufacturing container 20 is pressurized, themanufacturing container is sealed (see FIG. 7(b)). Specifically, byusing the gas 3, purging is performed in the void portion 11 of thecontainer body 21 into which the aqueous liquid 10 is injected, and thenthe lid 22 is fastened to the opening portion (vial mouth) of thecontainer body 21. In this way, the aqueous liquid 10 and the gas 3 aresealed in the manufacturing container 20.

Then, a syringe filled with the gas 3 is prepared, and an injectionneedle of the syringe is pierced into the rubber stopper 221.Thereafter, the gas 3 is further added into the manufacturing container20 from the syringe, thereby pressurizing the interior of themanufacturing container 20. Subsequently, the injection needle is pulledout of the rubber stopper 221. In this way, it is possible to obtain themanufacturing container 20 which is sealed in a state where the interiorof the manufacturing container 20 is pressurized due to the gas 3.

In the method for manufacturing bubbles of the present embodiment, theinternal pressure of the manufacturing container 20 (pressure of gas 3with which the void portion 11 is filled) is set to be higher than 1.0atm. Particularly, the internal pressure of the manufacturing container20 is preferably 1.5 to 10 atm, and more preferably 2 to 5 atm. In thisway, a portion of the gas 3 is micro-dispersed or dissolved in theaqueous liquid 10.

In a case where the gas 3 is micro-dispersed or dissolved in the aqueousliquid 10, when shock waves occur due to the collision between theaqueous liquid 10 and the manufacturing container 20 in Step (S4),bubbles 1 are easily generated. In this way, in Step (S4), more bubbles1 can be generated in the aqueous liquid 10.

In a case where the internal pressure of the manufacturing container 20is set to be a certain value higher than 1.0 atm, it is possible to moreeasily adjust the diameter and content of the bubbles 1 generated in theaqueous liquid 10.

In a case where Step (S4) and Step (S5) or Steps (S4) to (S6) areperformed in the same manner as in the first embodiment described aboveby using the manufacturing container 20 in which the aqueous liquid 10and the gas 3 are sealed as described above, a large amount of bubbles 1having a uniform size can be stably manufactured in the manufacturingcontainer 20. In addition, the manufacturing container 20 containing thelarge amount of bubbles 1 having the uniform size is obtained.

In the method for manufacturing bubbles of the present embodiment, themanufacturing container 20 in which the void portion 11 is pressurizeddue to the gas 3 is used. Therefore, at the stage in which themanufacturing container 20 is not yet vibrated, the gas 3 issufficiently micro-dispersed or dissolved in the aqueous liquid 10.Accordingly, when the manufacturing container 20 is vibrated in Step(S4), the bubbles 1 can be easily generated in the aqueous liquid 10,and the large amount of bubbles 1 having the uniform size can be moreeasily manufactured than in the method for manufacturing bubbles of thefirst embodiment described above.

The interior of the bubble-containing container (manufacturing container20) obtained in the present embodiment is pressurized. In a case wherethe internal pressure of the bubble-containing container is rapidlyreduced, the pressure reduction is likely to exert a negative influencesuch as a change in the particle size of the bubbles 1 in thebubble-containing liquid or a reduction in the content of the bubbles.Therefore, when the bubble-containing liquid is aspirated from theinterior of the bubble-containing container, it is preferable to reducethe internal pressure of the bubble-containing container in advance downto the atmospheric pressure.

For example, a syringe (syringe for pressure reduction), which isdifferent from the syringe (syringe for bubble aspiration) foraspirating the bubble-containing liquid from the bubble-containingcontainer, is prepared, and an injection needle of the syringe ispierced into the rubber stopper 221. At this time, an attention needs tobe paid to prevent the injection needle of the syringe for pressurereduction from not contacting with the bubble-containing liquid. Then,the gas 3 in the bubble-containing container is aspirated by operating aplunger of the syringe for pressure reduction, thereby reducing theinternal pressure of the bubble-containing container down to theatmospheric pressure. Thereafter, an injection needle of the syringe forbubble aspiration is pierced into the rubber stopper 221, and then thebubble-containing liquid is aspirated. At this time, it is preferable tomake the plunger of the syringe for pressure reduction in a state ofbeing pulled out of an external cylinder thereof. In this case, theinjection needle of the syringe for pressure reduction is opened to theinterior and the exterior of the bubble-containing container, and hencethe air freely comes into and out of the bubble-containing containerfrom the injection needle. With this constitution, when thebubble-containing liquid is aspirated, the internal pressure of thebubble-containing container is prevented from becoming negative, thatis, the internal pressure of the bubble-containing container is kept atthe atmospheric pressure, and hence the aforementioned negativeinfluence is not exerted on the generated bubbles 1.

In a state where the internal pressure of the bubble-containingcontainer is reduced down to the atmospheric pressure, the lid 22 may beremoved, and the bubble-containing liquid may be aspirated from theinterior of the bubble-containing container. In this case, because theinternal pressure of the bubble-containing container has been reduceddown to the atmospheric pressure, it is possible to reliably prevent thebubble-containing liquid from spurting out of the manufacturingcontainer 20 at the moment when the lid 22 is removed.

With the method for manufacturing bubbles and the bubble manufacturingcontainer of the second embodiment, the same operations and effects asin the method for manufacturing bubbles of the first embodiment are alsoobtained.

Third Embodiment

Next, a third embodiment of the method for manufacturing bubbles of thepresent invention will be described. The bubble 1 shown in FIG. 2(a)described above can be manufactured by the method for manufacturingbubbles of the present embodiment.

Hereinafter, regarding the method for manufacturing bubbles of the thirdembodiment, the differences between the methods for manufacturingbubbles of the first and second embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

In the present embodiment, the aqueous liquid 10 contains a material(outer shell material), constituting the outer shell 2 of the bubble 1,and an aqueous medium. That is, the method for manufacturing bubbles ofthe present embodiment is the same as the method for manufacturingbubbles of the second embodiment described above, except that theaqueous liquid 10 prepared in Step (S1) in the second embodimentcontains the outer shell material in addition to the aqueous medium.

[S1] Preparation Step

In the present embodiment, the outer shell material, which constitutesthe outer shell 2 of the bubble 1, and the aqueous medium are put intoan aqueous liquid preparation container (hereinafter, simply referred toas “preparation container”), and the outer shell material is dissolvedin the aqueous medium, thereby preparing the aqueous liquid 10. That is,the outer shell material and the aqueous medium are put into thepreparation container in a predetermined amount and then stirred suchthat the outer shell material is dissolved in the aqueous medium. Theorder of putting the outer shell material and the aqueous medium intothe preparation container is not particularly limited. As a method fordissolving the outer shell material in the aqueous medium, for example,stirring using a stirrer, an ultrasonic treatment, and the like can beused.

As the outer shell material, the amphipathic material described above isused. As the aqueous medium, the same aqueous medium as in the firstembodiment described above can be used.

In Step (S4), a content of the outer shell material in the aqueousliquid 10 is not particularly limited as long as the bubbles 1 can beformed in the aqueous liquid 10. The preferred content of the outershell material varies with the combination of the types of the outershell material and the aqueous medium. It is preferable that the outershell material is contained in the aqueous liquid 10 such that theconcentration of the material becomes equal to or higher than a criticalmicelle concentration (CMC). Specifically, the content of the outershell material contained in the aqueous liquid 10 is preferably 0.01 to50 wt %, and more preferably 0.1 to 20 wt %.

In this way, the concentration of the outer shell material in theaqueous liquid 10 more reliably becomes equal to or higher than thecritical micelle concentration, and hence the outer shell 2 (a liposomeor a micelle) can be reliably formed in the aqueous liquid 10.Therefore, in Step (S4) which will be described later, the gas 3 isincorporated into the liposome or the micelle in a simple manner, andhence the bubbles 1 having an intended size can be easily generated inthe aqueous liquid 10. Due to the existence of the outer shell 2, for along period of time, the bubbles 1 generated in the present embodimentcan prevent the gas 3 in the bubbles 1 from being eluted into theaqueous liquid 10 (aqueous medium). Consequently, the stability of thebubbles 1 is improved, and hence the accidental bursting of the bubbles1 can be more reliably prevented. Furthermore, the variation in the sizeof the generated bubbles 1 can be reduced. That is, the bubbles 1 havingthe uniform size can be generated.

The content of the aqueous medium in the prepared aqueous liquid 10 ispreferably 50 to 99.99 wt %, and more preferably 80 to 99.0 wt %. Inthis way, the outer shell material can be sufficiently dissolved in theaqueous medium, and hence a more homogeneous aqueous liquid 10 can beobtained.

Then, the same manufacturing container 20 as in the first and secondembodiments described above is prepared.

The aforementioned preparation container and the manufacturing container20 may be the same as or different from each other.

In a case where the preparation container and the manufacturingcontainer 20 are different from each other, for example, a containerhaving a relatively large volume can be used as the preparationcontainer, and a container having a relatively small volume can be usedas the manufacturing container 20 (container body 21). In this case, bypreparing a large amount of aqueous liquid 10 having a uniformcomposition in the preparation container, and dividing the aqueousliquid 10 into a plurality of manufacturing containers 20, the size(diameter) and amount of the bubbles generated in each of themanufacturing containers 20 can be made uniform.

In a case where the preparation container and the manufacturingcontainer 20 are the same as each other, Step (S2) can be skipped, andthis is advantageous because the process can be simplified.

In the present embodiment, as the preparation container, a containerdifferent from the manufacturing container 20 is used.

Then, by performing Steps (S2) to (S5) or Steps (S2) to (S6) in the samemanner as in the first embodiment described above, the large amount ofbubbles 1 having the uniform size can be stably manufactured in themanufacturing container 20. In addition, the manufacturing container 20containing the large amount of bubbles 1 having the uniform size isobtained.

In the present embodiment, due to the pressure of the shock waves thatoccur when the aqueous liquid 10 collides with the manufacturingcontainer 20 in Step (S4), the gas 3 is micro-dispersed or dissolved inthe aqueous liquid 10, and the outer shell material in the aqueousliquid 10 is changed to the bubble 1. The bubble 1 (outer shell 2)contains the gas 3, which is micro-dispersed or dissolved in the aqueousliquid 10 in Step (S3), and the gas 3 which is micro-dispersed ordissolved in the aqueous liquid 10 due to the vibration of this step.

In Step (S3), the interior of the manufacturing container 20 may not bepressurized (that is, the internal pressure of the manufacturingcontainer 20 is the atmospheric pressure) in the same manner as in thefirst embodiment or may be pressured in the same manner as in the secondembodiment.

With the method for manufacturing bubbles and the bubble manufacturingcontainer of the third embodiment, the same operations and effects as inthe methods for manufacturing bubbles of the first and secondembodiments are also obtained.

Fourth Embodiment

Next, a fourth embodiment of the method for manufacturing bubbles of thepresent invention will be described. The bubbles 1 shown in FIGS. 2(b)and 2(c) described above can be manufactured by the method formanufacturing bubbles of the present embodiment.

Hereinafter, regarding the method for manufacturing bubbles of thefourth embodiment, the differences between the methods for manufacturingbubbles of the first to third embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

In the present embodiment, the aqueous liquid 10 contains an aqueousmedium, an outer shell material, and a drug (medicine) 4. That is, themethod for manufacturing bubbles of the present embodiment is the sameas the method for manufacturing bubbles of the third embodimentdescribed above, except that the aqueous liquid 10 prepared in Step (S1)in the third embodiment contains the drug 4 in addition to the outershell material and the aqueous medium.

[S1] Preparation Step

In the present embodiment, the outer shell material, the drug 4, and theaqueous medium are put into the preparation container, and the outershell material and the drug 4 are dissolved in the aqueous medium,thereby preparing the aqueous liquid 10. That is, the outer shellmaterial, the drug 4, and the aqueous medium are put into thepreparation container in a predetermined amount and then stirred,thereby dissolving the outer shell material and the drug 4 in theaqueous medium. The order of putting the outer shell material, the drug4, and the aqueous medium into the preparation container is notparticularly limited. As a method for dissolving the outer shellmaterial and the drug 4 in the aqueous medium, for example, stirringusing a stirrer, an ultrasonic treatment, and the like can be used.

As the drug 4, the gene, the drug, and the like described above areused. A content of the drug 4 contained in the prepared aqueous liquid10 is preferably 0.1 to 50 wt %, and more preferably 20 to 50 wt %. Inthis way, a sufficient amount of drug 4 can be incorporated into themanufactured bubbles 1. As a result, it is possible to manufacture thebubbles 1 that are excellently effective for treating an affected site.In a case where the drug 4 constitutes the outer shell 2 instead of theouter shell material, the aqueous liquid 10 may not contain the outershell material.

In a case where Steps (S2) to (S5) or Steps (S2) to (S6) are performedin the same manner as in the third embodiment described above by usingthe aqueous liquid 10 prepared as above, the large amount of the bubbles1 (see FIGS. 2(b) and 2(c)) having the uniform size can be stablymanufactured in the manufacturing container 20. In addition, themanufacturing container 20 containing the large amount of the bubbles 1having the uniform size is obtained.

In the present embodiment, the bubble 1 (outer shell 2) contains the gas3 which is micro-dispersed or dissolved in the aqueous liquid 10 in Step(S3), the gas 3 which is micro-dispersed or dissolved in the aqueousliquid 10 due to the vibration in Step (S4), and the drug 4. As aresult, in the generated bubble 1, the drug 4 is sealed in the outershell 2 together with the gas 3 or incorporated into or adsorbed ontothe outer shell 2 itself.

3. How to Use

The bubble-containing container obtained as above is used for ultrasoundtherapy provided to patients or an ultrasonic diagnosis.

Specifically, first, an injection needle of a syringe is pierced intothe rubber stopper 221 of the lid 22. Then, by using the syringe, thebubble-containing liquid is aspirated from the interior of thebubble-containing container. The injection needle of the syringe intowhich the bubble-containing liquid is aspirated is pierced into a bloodvessel (for example, a vein) of a patient, and the bubble-containingliquid is injected into the blood vessel. In this way, the bubbles 1 aretransported to an affected site through the blood flow.

At the time of the ultrasound therapy, when the bubbles 1 reach thevicinity of the affected site, the bubbles are irradiated withtherapeutic ultrasonic waves having a frequency and an intensity atwhich the outer shell 2 may burst, thereby bursting the outer shell 2.In this way, by intensively supplying (applying) the drug 4 in thebubbles 1 to the affected site, the affected site can be treated.

In this case, it is also effective to perform the ultrasound therapy andthe ultrasonic diagnosis in combination. Specifically, the bubbles 1 inthe blood vessel are irradiated with the ultrasonic waves for diagnosis,and the reflection waves are monitored. In this way, the position or thebehavior of the bubbles 1 in the blood vessel (body) can be reliablyascertained. When the bubbles 1 reach the vicinity of the affected siteof interest, the bubbles are irradiated with the therapeutic ultrasonicwaves such that the bubbles 1 (outer shell 2) bursts. In this way, theaffected site can be treated by more accurately supplying the drugthereto.

In a case where a component (for example, carbon dioxide) showing highsolubility in the aqueous medium described above is used as the gas 3,after an elapse of a predetermined time from the manufacturing of thebubbles 1, the gas 3 in the outer shell 2 is eluted into the aqueousmedium. In a case where the gas 3 in the outer shell 2 is thencompletely eluted into the aqueous medium, the drug 4 becomes the onlycomponent sealed in the outer shell 2. That is, in this case, thebubbles 1 become liposomes or micelles in which only the drug 4 issealed in the outer shell 2. The liposomes or the micelles obtained inthis way can be used as a medical agent.

The intensity (power) of the therapeutic ultrasonic waves is preferablyabout 0.1 to 30 W/cm², and more preferably about 0.5 to 10 W/cm². In acase where the intensity of the therapeutic ultrasonic waves is withinthe aforementioned range, it is possible to more reliably burst thebubbles 1 and to eliminate or reduce the damage of normal cells aroundthe affected site. Furthermore, in a case where the intensity of thetherapeutic ultrasonic waves is within the aforementioned range, theirradiation time is preferably about 10 to 120 seconds, and morepreferably about 30 to 60 seconds.

The frequency of the ultrasonic waves radiated at the time of theultrasound therapy is preferably about 100 kHz to 10 MHz, and morepreferably about 700 kHz to 1 MHz. In a case where the frequency of theultrasonic waves radiated is within the aforementioned range, thebubbles can be burst by the ultrasonic waves of lower power.

Even in a case where the method for manufacturing bubbles of the fourthembodiment is used, the same operations and effects as in the methodsfor manufacturing bubbles of the first to thirds embodiments areobtained.

Fifth Embodiment

Next, a fifth embodiment of the method for manufacturing bubbles of thepresent invention will be described.

FIG. 8 is a flow chart for illustrating the fifth embodiment of themethod for manufacturing bubbles of the present invention.

Hereinafter, regarding the method for manufacturing bubbles of the fifthembodiment, the differences between the methods for manufacturingbubbles of the first to fourth embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

As shown in FIG. 8, the method for manufacturing bubbles of the presentembodiment includes Step (S7) and Step (S8) after Steps (S1) to (S5) (orSteps (S1) to (S6)) in the second embodiment described above. Step (S7)is a step of changing the internal pressure of the manufacturingcontainer. Step (S8) is a step of setting the number of revolution atwhich the manufacturing container is vibrated.

In a case where the number of revolution is not changed in Step (S8) (ina case where “NO” is selected in Step (S8) in FIG. 8), the method formanufacturing bubbles of the present embodiment further includes Step(S4′) and Step (S5′). In contrast, in a case where the number ofrevolution is changed in Step (S8) (in a case where “YES” is selected inStep (S8) in FIG. 8), the method for manufacturing bubbles of thepresent embodiment further includes Step (S9) and Step (S10).Furthermore, the method for manufacturing bubbles of the presentembodiment includes Step (S11) of changing the internal pressure of themanufacturing container. Hereinafter, each step following Step (S7) willbe sequentially described.

In the present embodiment, the aqueous liquid 10 may be composed only ofthe aqueous medium as in the first and second embodiments, may becomposed only of the aqueous medium and the outer shell material as inthe third embodiment, or may contain the aqueous medium, the outer shellmaterial, and the drug 4 as in the fourth embodiment.

[S7] Step of Changing Internal Pressure of Manufacturing Container

The internal pressure of the bubble-containing container (manufacturingcontainer 20) having undergone Step (S5) in the first embodimentdescribed above is changed.

(1) Case where Internal Pressure of Manufacturing Container is MadeHigher than Pressure in Step (S3)

The interior of the manufacturing container 20 is pressurized in thesame manner as in the aforementioned Step (S3). The gas 3 to be injectedmay be the same as or different from the gas 3 used in Step (S3).However, from the viewpoint of the stability of the finally generatedbubbles 1, it is preferable to use the same gas 3.

In such a manufacturing container 20, the interior of the manufacturingcontainer 20 is additionally pressurized by the internal pressure of themanufacturing container 20 in Step (S3). Therefore, the amount of thegas 3 which is micro-dispersed or dissolved in the aqueous liquid 10becomes larger than the amount of the gas 3 which is micro-dispersed ordissolved in the aqueous liquid 10 in Step (S3). Consequently, when themanufacturing container 20 is vibrated again in Step (S4′) or Step (S9)which will be described later, the gas 3 in the aqueous liquid 10 iseasily incorporated into the generated bubbles 1, and as a result, theamount of the generated bubbles 1 increases. Furthermore, a pressurehigher than the pressure applied to the bubbles 1 generated in Step (S4)is applied to the aqueous liquid 10. As a result, the bubbles 1 in theprocess generation are compressed under a higher pressure, and hence thediameter of the bubble 1 is easily reduced. Accordingly, it is possibleto generate the bubbles 1 having a diameter smaller than that of thebubbles 1 generated in Step (S4).

In this case, the internal pressure of the manufacturing container 20 ishigher than the pressure in Step (S3) preferably by 0.5 atm or more, andmore preferably by 1 to 10 atm. In this way, it is possible to morereliably generate the bubbles 1 having a diameter smaller than that ofthe bubbles 1 generated in Step (S4) described above.

(2) Case where Internal Pressure of Manufacturing Container is MadeHigher than 1.0 Atm but Lower than Pressure in Step (S3)

First, an empty syringe is prepared, and then an injection needle of thesyringe is pierced into the rubber stopper 221. Thereafter, the gas 3 inthe manufacturing container 20 is aspirated into the syringe. In thisway, the internal pressure of the manufacturing container 20 is reduced.Then, the injection needle is pulled out of the rubber stopper 221.

In the manufacturing container 20, when the manufacturing container 20is vibrated again in Step (S4′) or Step (S9) which will be describedlater, the pressure applied to the aqueous liquid 10 is lower than thepressure applied to the aqueous liquid 10 generated in Step (S4). As aresult, the bubbles 1 in the process of generation are compressed less,and hence the size of the bubbles 1 easily increases. Therefore, it ispossible to generate the bubbles 1 having a diameter larger than that ofthe bubbles 1 generated in Step (S4).

In this case, the internal pressure of the manufacturing container 20 isappropriately adjusted within such a range that the internal pressurebecomes higher than 1.0 atm but is lower than the pressure in Step (S3).

[S8] Step of Setting Number of Revolution at which ManufacturingContainer is Vibrated

After the internal pressure of the manufacturing container 20 is changedas described above, the number of revolution at which the container isvibrated again is set.

As in Step (S4) described above, the number of revolution at the time ofvibrating again the container is set to be equal to or higher than 5,000rpm.

In Step (S8), in a case where the number of revolution at the time ofvibrating again the container is not changed from the number ofrevolution in Step (S4), that is, in a case where “NO” is selected inStep (S8) in FIG. 8, the following Step (S4′) is performed.

On the other hand, in Step (S8), in a case where the number ofrevolution at the time of vibrating again the container is changed fromthe number of revolution in Step (S4), that is, in a case where “YES” isselected in Step (S8) in FIG. 8, the following Step (S9) is performed.

[S4′] Step of Vibrating Again Manufacturing Container

In the manner described above, the manufacturing container 20 whoseinternal pressure is changed is vibrated again at the same number ofrevolution as in Step (S4) described above. In this way, the bubbles 1having a diameter different from that of the bubbles 1 generated in Step(S4) are generated in the aqueous liquid 10.

In this step, the manufacturing container 20 is vibrated again at thesame number of revolution as in Step (S4). Therefore, the diameter ofthe bubbles 1 newly generated in the present embodiment changesaccording to the pressure changed in Step (S7). That is, by changing thepressure, the diameter of the bubbles 1 can be adjusted, and hence thebubbles 1 having different intended diameters (average diameters) can bemanufactured with excellent reproducibility. Furthermore, because thesetting of a device vibrating the manufacturing container 20 does notneed to be changed, the bubbles 1 having the different diameters can bemanufactured in a simpler manner.

[S5′] Step of Allowing Manufacturing Container to Stand

After the manufacturing container 20 is vibrated under theaforementioned condition, the manufacturing container 20 is allowed tostand in the same manner as in Step (S5). In this way, the large amountof bubbles 1 having the different sizes can be stably manufactured inthe manufacturing container 20. In addition, the manufacturing container20 (bubble-containing container) containing the large amount of bubbles1 described above is obtained.

After the container is allowed to stand, Step (S11) is performed.

[S9] Step of Vibrating Again Manufacturing Container

The manufacturing container 20 whose internal pressure is changed isvibrated again at the number of revolution different from that in Step(S4) described above. In this way, the bubbles 1 having a diameterdifferent from that of the bubbles 1 generated in Step (S4) aregenerated in the aqueous liquid 10.

(1) Case where Manufacturing Container is Vibrated Again at Number ofRevolution Higher than that in Step (S4)

The manufacturing container 20 is vibrated again in the same manner asin Step (S4) described above, except that the number of revolution atwhich the manufacturing container 20 is vibrated is made higher than thenumber of revolution in Step (S4).

In this case, the number of revolution of the manufacturing container 20is not particularly limited as long as it is higher than the number ofrevolution in Step (S4). The number of revolution in this case ispreferably 6,000 to 20,000 rpm, and more preferably 7,000 to 20,000 rpm.In this way, because the manufacturing container 20 is vibrated at thenumber of revolution higher than that in Step (S4), it is possible togenerate the bubbles 1 having a diameter smaller than that of thebubbles 1 generated in Step (S4). Furthermore, by setting the number ofrevolution of the manufacturing container 20 within the aforementionedrange, it is possible to more reliably prevent the bubbles 1 generatedin Step (S4) and the present step from being destroyed due to collisionor from coarsening by being combined with each other. As a result, it ispossible to manufacture the bubbles 1 generated in Step (S4) and thebubbles 1 having a diameter smaller than that of the bubbles 1 generatedin Step (S4).

(2) Case where Manufacturing Container is Vibrated Again at Number ofRevolution Lower than that in Step (S4)

The manufacturing container 20 is vibrated again in the same manner asin Step (S4), except that the number of revolution at which themanufacturing container 20 is vibrated is made lower than that in Step(S4).

In this case, the number of revolution of the manufacturing container 20is not particularly limited as long as it is lower than the number ofrevolution in Step (S4). The number of revolution in this case ispreferably 5,000 to 9,000 rpm, and more preferably 5,500 to 7,500 rpm.In this way, the manufacturing container 20 is vibrated at the number ofrevolution lower than that in Step (S4), and hence the bubbles 1 havinga diameter larger than that of the bubbles 1 generated in Step (S4) canbe generated. Furthermore, by setting the number of revolution of themanufacturing container 20 to be within the aforementioned range, it ispossible to more reliably prevent the bubbles 1 generated in Step (S4)and the present step from being destroyed due to the collision or fromcoarsening by being combined with each other. As a result, it ispossible to manufacture the bubbles 1 generated in Step (S4) and thebubbles 1 having a diameter larger than that of the bubbles 1 generatedin Step (S4).

In this step, the manufacturing container 20 is vibrated again at thenumber of revolution different from that in Step (S4). Therefore, thediameter of the bubbles 1 newly generated in the present embodimentchanges according to the change of the internal pressure of themanufacturing container 20 and to the number of revolution at the timeof vibrating again the container. In this way, by changing both theinternal pressure of the manufacturing container 20 and the number ofrevolution at the time of vibrating again the container, it is possibleto generate the bubbles 1 having a diameter greatly different from thediameter obtained in the first embodiment described above. Accordingly,in a case where the bubbles 1 that greatly differ from each other interms of the average diameter are manufactured, the present step isadvantageous.

[S10] Step of Allowing Manufacturing Container to Stand

After the manufacturing container 20 is vibrated in Step (S9), themanufacturing container 20 is allowed to stand in the same manner as inStep (S5). In this way, the bubbles 1 having the different diameters canbe stably manufactured in the manufacturing container 20. In addition,the manufacturing container 20 (bubble-containing container) containingthe large amount of bubbles 1 described above is obtained.

After the container is allowed to stand, Step (S11) is performed.

[S11] Step of Changing Again Internal Pressure of ManufacturingContainer

In a case where the internal pressure of the manufacturing container 20is not changed, that is, in a case where “NO” is selected in Step (S11)in FIG. 8, the method for manufacturing bubbles of the presentembodiment is finished. In this way, first bubbles 1 and second bubbles1 having different average diameters within a range of 10 nm to 1,000 μmare manufactured.

In contrast, in a case where the internal pressure of the manufacturingcontainer 20 is changed, that is, in a case where “YES” is selected inStep (S11) in FIG. 8, Step (S8) is performed. Then, Steps (S4′), (S5′),and (S11) described above or Steps (S9), (S10), and (S11) are repeated.In this way, it is possible to manufacture the bubbles 1 having aplurality of different average diameters within a range of 10 nm to1,000 μm.

In a case where the pressure is repeatedly changed in Step (S11), it ispossible to manufacture the bubbles 1 having the plurality of differentaverage diameters according to the number of times the pressure ischanged.

The bubble-containing container obtained as above contains the bubbles 1having the different diameters in the aqueous liquid 10. The ease ofpassing the bubbles 1 in blood vessels and the site to which the bubblesare transported vary with the difference in the size (for example, thesmaller the size of the bubbles 1 is, the farther the bubbles 1 can betransported toward the end of a capillary). Therefore, thebubble-containing liquid obtained as above can be used in many waysaccording to the purpose of the ultrasound therapy.

With the method for manufacturing bubbles of the fifth embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to fourth embodiments are also obtained.

Sixth Embodiment

Next, a sixth embodiment of the method for manufacturing bubbles of thepresent invention will be described.

FIG. 9 is a partial cross-sectional view showing the vicinity of a lidof a manufacturing container used in the sixth embodiment of the methodfor manufacturing bubbles of the present invention.

In the following description, the upper side in FIG. 9 will be referredto as “top”, and the lower side in FIG. 9 will be referred to as“bottom”.

Hereinafter, regarding the method for manufacturing bubbles of the sixthembodiment, the differences between the methods for manufacturingbubbles of the first to fifth embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the methods for manufacturing bubbles of the first to fifthembodiments described above, except that the constitution of the lid 22of the manufacturing container 20 (the second embodiment of the bubblemanufacturing container) is different.

The lid 22 shown in FIG. 9 includes, in addition to the rubber stopper221 and the fastening portion 222 described above, a bottom plateportion 223 which adheres to the bottom surface of the rubber stopper221 through an adhesive or the like. In other words, the bottom plateportion 223 is provided in the opening portion of the container body 21in a state of adhering to the rubber stopper 221. Because themanufacturing container 20 of the present embodiment includes the bottomplate portion 223, the mass of the lid 22 of the present embodimentbecomes larger than that of the lid 22 of the manufacturing container 20of the first embodiment described above. That is, the bottom plateportion 22 becomes a weight portion that increases the mass of themanufacturing container 20.

The bottom plate portion 223 is a disk-like member having a diametersmaller than that of the rubber stopper 221. Furthermore, when seen in aplan view, a through hole 224, into which an injection needle of asyringe is inserted, is formed approximately at the center of the bottomplate portion 223 so as to correspond to the opening of the fasteningportion 222. The size of the through hole 224 is not particularlylimited. In a case where the through hole 224 has the same size as therubber stopper 221 exposed from the fastening portion 222, in Step (S3),the injection needle can be pierced into anywhere within a region of therubber stopper 221 exposed from the fastening portion 222. Furthermore,as shown in FIG. 9, the through hole 224 may have a size that enablesthe injection needle to be inserted thereinto. In this case, a mark (notshown in the drawing) may be made on the top surface of the rubberstopper 221 in a position corresponding to the through hole 224, and theinjection needle may be pierced into the rubber stopper 221 in themarked position.

The bottom plate portion 223 may be provided with a plurality of throughholes 224. For example, the bottom plate portion 223 may be providedwith two through holes 224 including a through hole into which aninjection needle of a syringe filled with the gas 3 and an injectionneedle of the aforementioned pressure reduction syringe are inserted anda through hole into which an injection needle of a bubble aspiratingsyringe is inserted. In this constitution, by using the pressurereduction syringe, in a state where the internal pressure of thebubble-containing container is set to be atmospheric pressure, thebubbles 1 can be aspirated into the bubble aspirating syringe.Therefore, it is possible to more reliably prevent the occurrence ofnegative influences such as the change in the particle size of thebubbles 1 in the aspirated bubble-containing liquid or the reduction ofthe content. Furthermore, the bottom plate portion 223 may be providedwith three through holes 224 into which the injection needle of each ofthe syringes is inserted. In addition, on the top surface of the rubberstopper 221, a mark at which the injection needle of each of thesyringes is pierced may be made in a position corresponding to eachthrough hole 224.

Examples of a material constituting the bottom plate portion 223 includevarious ceramic materials and metal materials. Among these, the materialhaving a density (equal to or higher than 2,000 kg/m³) equal to orhigher than the density of glass is preferable. Examples of such amaterial include stainless steel such as cast iron (density: about 7,000to 7,700 kg/m³), 18/8 chromium nickel steel (density: about 7,900kg/m³), and V2A steel (density: about 7,900 kg/m³), aluminum (density:about 2,700 kg/m³), duralumin (density: about 2,700 kg/m³), lead(density: about 11,340 kg/m³), iron (density: about 7,870 kg/m³), copper(density: about 8,900 kg/m³), brass (density: about 8,250 to 8,500kg/m³), nickel (density: about 8,350 kg/m³), cast iron (density: about7,000 to 7,700 kg/m³), zinc (density: about 7,130 kg/m³), tin (density:about 7,280 kg/m³), and the like. Among these, one kind of material canbe used singly, or two or more kinds of materials may be used incombination. Among these materials, iron or an iron alloy such asstainless steel is particularly preferably used, because these materialshave high specific gravity and exhibit high corrosion resistance withrespect to the components constituting the aqueous liquid 10.

The mass of the bottom plate portion 223 constituted with theaforementioned material is greater than the mass of a member formed of amaterial having low specific gravity (density) such as synthetic rubberor a resin. By increasing the mass of the bottom plate portion 223, inStep (S4), it is possible to further increase the magnitude of the shockwaves which occur when the aqueous liquid 10 collies with the topsurface (bottom plate portion 223) of the manufacturing container 20. Asa result, fine bubbles 1 can be more easily and stably generated in theaqueous liquid 10.

In a case where the lid 22 is used, in Step (S3), the injection needleof the syringe filled with the gas 3 is pierced into the rubber stopper221 such that the needle is inserted into the through hole 224 of thebottom plate portion 223. Then, by further adding the gas 3 into themanufacturing container 20 from the syringe in the same manner as in thesecond embodiment described above, the interior of the manufacturingcontainer 20 is pressurized. Thereafter, by pulling the injection needleout of the lid 22 (rubber stopper 221), it is possible to obtain themanufacturing container 20 sealed in a state where the interior of themanufacturing container 20 is pressurized due to the gas 3.

By using the manufacturing container 20 (a bubble manufacturingcontainer of the present embodiment) constituted as above, abubble-containing container can be obtained through the same steps as inthe method for manufacturing bubbles of the first to fifth embodimentsdescribed above.

With the method for manufacturing bubbles of the sixth embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to fifth embodiments are also obtained.

Seventh Embodiment

Next, a seventh embodiment of the method for manufacturing bubbles ofthe present invention will be described.

FIGS. 10(a) to 10(f) are cross-sectional views schematically showingcontainers used in the seventh embodiment of the method formanufacturing bubbles of the present invention.

In the following description, the upper side in each of FIGS. 10(a) to10(f) will be referred to as “top”, and the lower side in each of FIGS.10(a) to 10(f) will be referred to as “bottom”.

Hereinafter, regarding the method for manufacturing bubbles of theseventh embodiment, the differences between the methods formanufacturing bubbles of the first to sixth embodiments and the methodfor manufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the methods for manufacturing bubbles of the first to sixthembodiments described above, except that the container (manufacturingcontainer) has a different shape.

In the present embodiment, the manufacturing container 20 (the thirdembodiment of the bubble manufacturing container) includes the containerbody 21 having various shapes shown in FIGS. 10(a) to 10(d) and a lid(not shown in the drawings) matching with the shape of the top surfaceof each container body 21. As shown in FIGS. 10(a) to 10(d), the innersurface of the container body 21 includes at least one of a convexsurface, a concave surface, and a corrugated surface.

The container body 21 shown in FIG. 10(a) is a container in which thetop and bottom surfaces thereof are in the form of the convex surfaceprojected toward the inside. The manufacturing container 20 shown inFIG. 10(b) is a container in which the top and bottom surfaces thereofare in the form of the corrugated surface. The manufacturing container20 shown in FIG. 10(c) is a container in which the top and bottomsurfaces thereof are in the form of the concave surface projected towardthe outside. The manufacturing container shown in FIG. 10 (d) is acontainer in which the lateral surface thereof is in the form of theconvex surface curving toward the inside of the manufacturing container20.

These surfaces (the concave surface, the convex surface, and thecorrugated surface) have a large surface area compared to a flatsurface. Therefore, in Step (S4) described above, the area in which theaqueous liquid 10 can collide with the inner surface of themanufacturing container increases, and hence more shock waves can occur.Furthermore, the magnitude of the pressure of the occurring shock wavesvaries with the shape of such surfaces. As a result, a large amount ofbubbles 1 having different diameters can be generated in the aqueousliquid 10.

As shown in FIG. 10(e), the height of the container body 21 can be madegreater than the height of the container body 21 of the first to sixthembodiments described above. In a case where such a container body 21 isused, when the manufacturing container is vibrated in Steps (S4), (S4′),and (S9), the aqueous liquid 10 moves a long distance, and hence themagnitude of the shock waves that occur at the time of collision can beincreased. As a result, the size of the bubbles 1 manufactured in theaqueous liquid 10 can be further reduced.

In contrast, as shown in FIG. 10(f), the height of the container body 21can be made smaller than the height of the container body 21 of thefirst to sixth embodiments described above. In a case where such acontainer body 21 is used, when the manufacturing container is vibratedin Steps (S4), (S4′), and (S9), the aqueous liquid 10 moves a shortdistance, and hence the number of times the aqueous liquid 10 collideswith the inner surface of the manufacturing container can be increased.Therefore, the pressure, resulting from the shock waves and applied tothe aqueous liquid 10, can be further increased. As a result, morebubbles 1 can be manufactured in the aqueous liquid 10.

With the method for manufacturing bubbles of the seventh embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to sixth embodiments are also obtained.

Eighth Embodiment

Next, an eighth embodiment of the method for manufacturing bubbles ofthe present invention will be described.

FIGS. 11(a) to (c) are perspective views for illustrating the eighthembodiment of the method for manufacturing bubbles of the presentinvention. FIG. 12 shows views for illustrating the constitution of thevicinity of a rubber stopper of a Mininert valve shown in FIG. 11(a) (ahandle is not shown). FIG. 12(a) is a top view of the vicinity of therubber stopper of the Mininert valve. FIG. 12(b) is a cross-sectionalview of FIG. 12(a) taken along the line X-X. FIG. 13 is across-sectional view of a bubble-containing container shown in FIG.11(c).

In the following description, the upper side in each of FIGS. 11(a) to11(c), FIG. 12(b), and FIG. 13 as well as the front side in FIG. 12(a)based on the paper surface will be referred to as “top”, and the lowerside in each of FIGS. 11(a) to (c), FIG. 12(b), and FIG. 13 as well asthe back side in FIG. 12(a) based on the paper surface will be referredto as “bottom”. Furthermore, the left side in each of FIGS. 11(a) to11(c) will be referred to as “left”, and the right side in each of FIGS.11(a) to 11(c) will be referred to as “right”.

Hereinafter, regarding the method for manufacturing bubbles of theeighth embodiment, the differences between the methods for manufacturingbubbles of the first to seventh embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the methods for manufacturing bubbles of the first to fifthembodiments described above, except that the manufacturing container hasa different constitution.

[S1] Preparation Step

The manufacturing container 20 (the fourth embodiment of the bubblemanufacturing container) shown in FIG. 11(a) is prepared.

In the present embodiment, the manufacturing container 20 includes thecontainer body 21 and the lid 22, similarly to the manufacturingcontainer 20 of the first embodiment described above. As shown in FIG.11(a), the lid 22 includes a Mininert valve 30 on the upper side thereofand a tube 33 connecting the rubber stopper 221 to the Mininert valve30.

The Mininert valve 30 includes a valve body 31 having a through hole311, a rubber stopper 32 in which a duct 321, being buried in thethrough hole 311 and penetrating the valve body 31 in a thicknessdirection (vertical direction), is formed, and a handle (an opening andclosing mechanism) 34 which controls the opening and closing of the duct321.

The valve body 31 has approximately a cuboid shape. In the vicinity ofthe center of the valve body 31 in the longitudinal direction thereof,the through hole 311 that penetrates the valve body 31 in the thicknessdirection (vertical direction) is formed (see FIGS. 12(a) and 12(b)).Furthermore, as shown in FIG. 12(b), in the valve body 31, a throughhole 312 that penetrates the valve body 31 in the longitudinal directionthereof is formed.

The rubber stopper 32 has approximately a cylindrical shape, and isconstituted with, for example, silicon rubber. The rubber stopper 32 isinserted into the through hole 311 of the valve body 31 and fixed to(buried in) the valve body 31. Approximately at the center of the rubberstopper 32, the duct 321 is formed into which an injection needle 41 ofa syringe 40 can be inserted. Furthermore, in the rubber stopper 32, athrough hole 322 is formed which is orthogonal to the duct 321 andpenetrates the rubber stopper 32 in a width direction (horizontaldirection) thereof (see FIG. 12(b)). The through hole 322 of the rubberstopper 32 and the through hole 312 of the valve body 31 are incommunication with each other, and a shaft 342 of a handle 34, whichwill be described later, are slidably inserted into the through hole 322and the through hole 312.

The handle 34 includes a pair of knobs 341 which is provided at both endsides of the valve body 31 in the longitudinal direction (horizontaldirection) thereof, and a shaft 342 which is connected to each of theknobs 341 and slidably inserted into the through hole 322 of the rubberstopper 32 and the through hole 312 of the valve body 31. In a portionof the shaft 342, a through hole 343 penetrating the portion in thethickness direction (vertical direction in FIG. 11(a)) is formed.

The tube 33 is not particularly limited, and constituted with, forexample, a tube made of silicon. The top end portion of the tube 33 isin communication with the duct 321 of the rubber stopper 32, and thebottom end portion of the tube 33 is in communication with the interiorspace (void portion 11) of the container body 21 through the rubberstopper 221. In other words, the duct 321 and the interior space of thecontainer body 21 are in communication with each other through the tube33.

In the manufacturing container 20 of the present embodiment, as shown inFIG. 11(a), the knob 341 on the left side is pushed to the right sidesuch that the knob 341 on the left side contacts a left end portion ofthe valve body 31. In this way, when seen in a plan view (top view), athrough hole 343 of the shaft 342 is superposed on (in communicationwith) the duct 321. As a result, the duct 321 is opened, and hence theduct 321, the tube 33, and the interior space of the container body 21become in communication with each other (see FIGS. 11(a) and 12(b)). Incontrast, as shown in FIG. 11(b), the knob 341 on the right side ispushed to the left such that the knob 341 on the right side contacts aright end portion of the valve body 31. In this way, when seen in theplan view (top view), a position of the through hole 343 of the shaft342 deviates from a position of the duct 321. As a result, the duct 321is closed, and the interior of the manufacturing container 20 is sealed.

In the present embodiment, similarly to the lid 22 shown in FIG. 9, thelid 22 includes the rubber stopper 221, the fastening portion 222, andthe bottom plate portion 223 (see FIG. 13). As shown in FIG. 13, abottom end portion (end portion on the side connected to the rubberstopper 221) of the tube 33 is disposed in a position corresponding tothe through hole 224 of the bottom plate portion 223. Therefore, thetube 33 is in communication with the interior of the container body 21.

[S3] Step of Sealing Manufacturing Container

By using the gas 3, purging is performed in the void portion 11 of thecontainer body 21 into which the aqueous liquid 10 is injected, and thenthe lid 22 is inserted into the opening portion (vial mouth) of thecontainer body 21. As a result, the aqueous liquid 10 and the gas 3 aresealed in the manufacturing container 20.

Then, the syringe 40 filled with the gas 3 is prepared. As shown in FIG.11(a), the knob 341 on the left side of the handle 34 is pushed to theright side, such that the duct 321 is opened and becomes incommunication with the tube 33. Thereafter, the injection needle 41 ofthe syringe 40 is inserted into the duct 321, and then through theMininert valve 30 and the tube 33, the gas 3 is further added into themanufacturing container 20 from the syringe 40. In this way, theinterior of the manufacturing container 20 is pressurized.

Thereafter, the injection needle 41 is pulled out of the duct 321, andthe knob 341 on the right side of the handle 34 is pushed to the leftside such that the duct 321 is closed, and in this way, the duct 321 andthe tube 33 are not in communication with each other.

Then, a thermal treatment is performed on a portion of the tube 33,thereby forming a sealing portion 331 in which the interior space of thetube 33 is closed (see FIG. 11(b)).

Subsequently, by cutting the tube 33 at the Mininert valve 30 side(upper side in the drawing) of the sealing portion 331, themanufacturing container 20 can be obtained which is sealed in a statewhere the interior of the manufacturing container 20 is pressurized dueto the gas 3 (see FIG. 11(c)).

By performing the following steps in the same manner as in the first tofifth embodiments described above, a large amount of bubbles 1 having auniform size can be stably manufactured in the manufacturing container20. In addition, the manufacturing container (sealed container) 20(bubble-containing container) containing the large amount of bubbles 1having the uniform size is obtained.

In the present embodiment, in Step (S3), the interior of themanufacturing container 20 can be pressurized without directly piercingthe injection needle 41 into the rubber stopper 221 of the lid 22. Thatis, in the present embodiment, because there is no through hole in thelid 22 and the tube 33 (the region from the rubber stopper 221 to thesealing portion 331), the sealing property of the interior of themanufacturing container 20 can be improved. By improving the sealingproperty of the interior of the manufacturing container 20, in thefinally obtained bubble-containing container, the bubbles 1 can morestably exist in the aqueous liquid 10. That is, the long-termstorability of the bubble-containing container is further improved.

With the method for manufacturing bubbles of the eighth embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to seventh embodiments are also obtained.

Ninth Embodiment

Next, a ninth embodiment of the method for manufacturing bubbles of thepresent invention will be described.

FIG. 14 is a perspective view for illustrating a manufacturing containerused in the ninth embodiment of the method for manufacturing bubbles ofthe present invention.

In the following description, the upper side in FIG. 14 will be referredto as “top”, and the lower side in FIG. 14 will be referred to as“bottom”. Furthermore, the left side in FIG. 14 will be referred to as“left”, and the right side in FIG. 14 will be referred to as “right”.

Hereinafter, regarding the method for manufacturing bubbles of the ninthembodiment, the differences between the methods for manufacturingbubbles of the first to eighth embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment isdifferent from the method for manufacturing bubbles of the eighthembodiment described above in that the Mininert valve 30 is provided onthe lateral surface of the container body 21.

As shown in FIG. 14, the manufacturing container 20 (the fifthembodiment of the bubble manufacturing container) used in the presentembodiment includes the container body 21, the lid 22, the Mininertvalve 30, and the tube 33 which connects the container body 21 to theMininert valve 30, which are used in the method for manufacturingbubbles of the eighth embodiment described above. Furthermore, in themanufacturing container 20 of the present embodiment, the Mininert valve30 is provided on the lid 22 side (upper side in the drawing) above thesurface of the aqueous liquid 10.

The tube 33 is constituted with a tube (pipe) made of glass andintegrally formed with the container body 21 (vial).

As shown in FIG. 14, a left end portion of the tube 33 is incommunication with the interior space (void portion 11) of the containerbody 21, and a right end portion of the tube 33 is in communication withthe duct 321 of the rubber stopper 32. Accordingly, in the presentembodiment, the duct 321 and the interior space of the container body 21are also in communication with each other through the tube 33.

By using the manufacturing container 20 constituted as above, abubble-containing container can be obtained through the same steps as inthe method for manufacturing bubbles of the eighth embodiment describedabove.

With the method for manufacturing bubbles of the ninth embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to eighth embodiments are also obtained.

Tenth Embodiment

Next, a tenth embodiment of the method for manufacturing bubbles of thepresent invention will be described.

FIG. 15 is a cross-sectional view for illustrating a manufacturingcontainer used in the tenth embodiment of the method for manufacturingbubbles of the present invention.

In the following description, the upper side in FIG. 15 will be referredto as “top”, and the lower side in FIG. 15 will be referred to as“bottom”. Furthermore, the left side in FIG. 15 will be referred to as“left”, and the right side in FIG. 15 will be referred to as “right”.

Hereinafter, regarding the method for manufacturing bubbles of the tenthembodiment, the differences between the methods for manufacturingbubbles of the first to ninth embodiments and the method formanufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The bubble manufacturing container of the present embodiment isconstituted such that the longitudinal direction thereof becomessubstantially a horizontal direction (in the first to ninth embodimentsdescribed above, the longitudinal direction of the manufacturingcontainer 20 is a vertical direction). In Step (S4), the manufacturingcontainer is vibrated in the horizontal direction. In this respect, themethod for manufacturing bubbles of the present embodiment is differentfrom the methods for manufacturing bubbles of the first to ninthembodiments described above. Specifically, in Step (S4), themanufacturing container 20 shown in FIG. 15 is vibrated such that itreciprocates substantially in the horizontal direction thereof.

[S1] Preparation Step

The manufacturing container 20 (the sixth embodiment of the bubblemanufacturing container) shown in FIG. 15 is prepared.

The manufacturing container 20 of the present embodiment includes thecontainer body 21 having a cylindrical portion 211 on the top portionthereof, a rubber stopper 23 sealing an opening of the cylindricalportion 211, and two weight portions 5 fixed to both end portions of thecontainer body 21.

The container body 21 has an approximately cylindrical shape that islong in the horizontal direction (right and left direction in FIG. 15).Approximately at the center of the container body 21, the cylindricalportion (projection portion) 211 is formed which projects in thevertical direction from the top surface of the container body 21. In thecontainer body 21 of the present embodiment, only the opening of thecylindrical portion 211 is opened to the outside. At both end sides ofthe container body 21, a screw groove 212 is formed.

The rubber stopper 23 is not particularly limited, and for example, arubber stopper made of silicon can be used.

The weight portions 5 each have a disk-like flat plate portion 51 and acylindrical portion 52 that stands on the edge of the flat plate portion51. When seen in a cross-sectional view, each of the weight portions 5looks like an approximately C-shaped member. On the innercircumferential side of the cylindrical portion 52, the screw groove 521is formed which can be screwed with the screw groove 212 of thecontainer body 21. By screwing the screw groove 521 of the weightportions 5 with the screw groove 212 of the container body 21, theweight portions 5 are mounted on (fixed to) the container body 21 in astate where the flat plate portion 51 adheres to each of the endportions of the container body 21.

Similarly to the bottom plate portion 223 of the lid 22 shown in FIG. 9described above, the weight portions 5 are each constituted with amaterial having a relatively high specific gravity, such as a ceramicmaterial and a metal material. Therefore, by mounting the weightportions 5 on the container body 21, the masses of both end portions ofthe container body 21 can be increased. In this way, in Step (S4), it ispossible to further increase the magnitude of the shock waves that occurwhen the aqueous liquid 10 collides with the portion (particularly, bothend portions) of the container body 21 fixed to the weight portions 5.As a result, fine bubbles 1 can be more easily and stably generated inthe aqueous liquid 10.

As the material constituting the weight portions 5, among metalmaterials, iron or an iron alloy such as stainless steel is particularlypreferable, because these materials have high specific gravity andexhibit high corrosion resistance with respect to the componentsconstituting the aqueous liquid 10.

The weight portions 5 can be easily detached from and attached to thecontainer body 21. By changing the constituent materials and/or the sizeof the weight portions 5, the masses of the weight portions 5 can beappropriately adjusted. By adjusting the masses of the weight portions5, in Step (S4), the size and amount of the bubbles 1 generated in theaqueous liquid 10 can be adjusted. That is, in the method formanufacturing bubbles of the present embodiment, the bubbles 1 ofvarious sizes and contents can be manufactured using the samemanufacturing container 20. Therefore, it is not necessary to prepareplural kinds of manufacturing containers 20 of different sizes accordingto the bubbles 1 of intended sizes and contents, and hence theproductivity of the bubble-containing container is improved.

[S3] Step of Sealing Manufacturing Container

By using the gas 3, purging is performed in the container body 21 intowhich the aqueous liquid 10 is injected. Then, the rubber stopper 23 isinserted into the opening of the cylindrical portion 211 of thecontainer body 21. In this way, the aqueous liquid 10 and the gas 3 aresealed in the manufacturing container 20.

Then, the syringe 40 filled with the gas 3 is prepared, and theinjection needle 41 of the syringe 40 is pierced into the rubber stopper23. Thereafter, the gas 3 is further added into the manufacturingcontainer 20 from the syringe 40. In this way, the interior of themanufacturing container 20 is pressurized. Then, by pulling theinjection needle out of the rubber stopper 23, it is possible to obtainthe manufacturing container 20 which is sealed in a state where theinterior of the manufacturing container 20 is pressurized due to the gas3.

[S4] Step of Vibrating Manufacturing Container

Then, the manufacturing container 20 is vibrated such that the aqueousliquid 10 repeatedly collides with the both end portions and the latersurface (particularly, the both end portions) of the manufacturingcontainer 20. In the present embodiment, the manufacturing container 20is vibrated such that it substantially reciprocates in the horizontaldirection (longitudinal direction) of the manufacturing container 20.

In the present embodiment, the manufacturing container 20 can bevibrated under the same condition as in Step (S4) in the firstembodiment described above.

By performing the following steps in the same manner as in the first tofifth embodiments described above, a large amount of bubbles 1 having auniform size can be stably manufactured in the manufacturing container20. In addition, the manufacturing container (sealed container) 20(bubble-containing container) containing the large amount of bubbles 1having the uniform size is obtained.

With the method for manufacturing bubbles of the tenth embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to ninth embodiments are also obtained.

Eleventh Embodiment

Next, an eleventh embodiment of the method for manufacturing bubbles ofthe present invention will be described.

FIG. 16 shows cross-sectional views for illustrating a manufacturingcontainer used in the eleventh embodiment of the method formanufacturing bubbles of the present invention. FIG. 16(a) shows themanufacturing container in a disassembled state, and FIG. 16(b) showsthe manufacturing container in an assembled state.

In the following description, the upper side in each of FIGS. 16(a) and16(b) will be referred to as “top”, and the lower side in each of FIGS.16(a) and 16(b) will be referred to as “bottom”. Furthermore, the leftside in each of FIGS. 16(a) and 16(b) will be referred to as “left”, andthe right side in each of FIGS. 16(a) and 16(b) will be referred to as“right”.

Hereinafter, regarding the method for manufacturing bubbles of theeleventh embodiment, the differences between the methods formanufacturing bubbles of the first to tenth embodiments and the methodfor manufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the method for manufacturing bubbles of the tenth embodimentdescribed above in which the manufacturing container 20 shown in FIG. 15is used, except that both ends of the container body 21 of themanufacturing container 20 (the seventh embodiment of the bubblemanufacturing container) are opened to the outside as shown in FIGS.16(a) and 16(b). That is, the container body 21 is constituted with acylindrical member whose both ends are opened to the outside.

In a case where the manufacturing container 20 constituted as above isused, first, by screwing the screw grooves 521 of the weight portions 5with the screw grooves 212 of the container body 21, the weight portions5 are mounted on the container body 21. As shown in FIG. 16(b), in astate where the weight portions 5 are mounted on the container body 21,the flat plate portion 51 adheres to each of the end portions of thecontainer body 21.

Between the flat plate portion 51 and each of the both end portions ofthe container body 21, a packing for improving the adhesion between theweight portions 5 and the container body 21 may be disposed, althoughthis constitution is not shown in the drawing. In this way, the sealingproperty of the manufacturing container 20 can be improved.

Then, through the same steps as in the method for manufacturing bubblesof the present embodiment described above, a bubble-containing containercan be obtained.

By piercing an injection needle of a syringe into the rubber stopper 23and then aspirating the bubble-containing liquid, the bubble-containingcontainer obtained as above can be used. In this constitution, bydetaching the weight portions 5 from the container body 21, it ispossible to directly take the bubble-containing liquid out of thebubble-containing container without using the syringe.

With the method for manufacturing bubbles of the eleventh embodiment,the same operations and effects as in the methods for manufacturingbubbles of the first to tenth embodiments are also obtained.

Twelfth Embodiment

Next, a twelfth embodiment of the method for manufacturing bubbles ofthe present invention will be described.

FIG. 17 is a cross-sectional view for illustrating a manufacturingcontainer used in the twelfth embodiment of the method for manufacturingbubbles of the present invention.

In the following description, the upper side in FIG. 17 will be referredto as “top”, and the lower side in FIG. 17 will be referred to as“bottom”.

Hereinafter, regarding the method for manufacturing bubbles of thetwelfth embodiment, the differences between the methods formanufacturing bubbles of the first to eleventh embodiments and themethod for manufacturing bubbles of the present embodiment will bemainly described, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the methods for manufacturing bubbles of the first to fifthembodiments described above, except that the manufacturing container hasa different constitution.

[S1] Preparation Step

The manufacturing container 20 (the eighth embodiment of the bubblemanufacturing container) shown in FIG. 17 is prepared.

The manufacturing container 20 of the present embodiment includes thecontainer body 21, the lid 22 sealing the container body 21, the weightportion 5 provided on the other end side of the container body 21 suchthat the weight portion 5 can move in the longitudinal direction of thecontainer body 21, and a pair of O-rings 6 fixing the weight portion 5.The lid 22 has the same constitution as the lid 22 of the manufacturingcontainer 20 of the first embodiment described above.

The container body 21 has an approximately bottomed cylindrical shape.The container body 21 includes a top body portion 215 on which the lid22 is mounted and a bottom body portion 216 on which the weight portion5 is mounted and which has an inner diameter smaller than that of thetop body portion 215. As shown in FIG. 17, the top body portion 215 has,on the bottom end portion thereof, a diameter-reduced portion 217reduced so as to become the same as the inner diameter of the bottombody portion 216. On an outer circumferential surface of one end side(top end side) of the top body portion 215, a screw groove 213 which canbe screwed with the fastening portion 222 (inner circumferential surfaceof the fastening portion 222) of the lid 22 is formed. Furthermore, onthe entirety of the outer circumferential surface of the bottom bodyportion 216, a screw groove 214 which can be screwed with the weightportion 5 (inner circumferential surface of the weight portion 5) isformed.

The material constituting the container body 21 is not particularlylimited, and various ceramic materials such as glass, a resin material,and the like can be used.

The length of the top body portion 215 in the longitudinal direction isnot particularly limited, but is preferably about 10 to 60 mm and morepreferably about 15 to 30 mm. The inner diameter of the top body portion215 is preferably about 5 to 20 mm, and more preferably about 8 to 15mm. The length of the bottom body portion 216 in the longitudinaldirection is not particularly limited, but is preferably about 10 to 35mm and more preferably about 13 to 23 mm. The inner diameter of thebottom body portion 216 is not particularly limited as long as it issmaller than the inner diameter of the top body portion 215. The innerdiameter of the bottom body portion 216 is preferably about 2 to 15 mm,and more preferably about 3 to 8 mm.

In a case where the container body 21 having such dimensions is used, anappropriate pressure is applied to the aqueous liquid 10 in the sealedspace within the container body 21. Therefore, the bubbles 1 having theuniform size can be stably obtained. Furthermore, in this case, at thetime of the ultrasonic diagnosis, since the bubble-containing liquid ina single manufacturing container 20 can be used up, it is possible toeliminate a waste of the manufactured bubble-containing liquid.

In a case where the inner diameter of the top body portion 215 and theinner diameter of the bottom body portion 216 are within theaforementioned range, a big difference occurs between the inner diameterof the top body portion 215 and the inner diameter of the bottom bodyportion 216, and the size of the diameter-reduced portion 217 increases.Accordingly, in Step (S4), when moving to the bottom body portion 216side from the top body portion 215, the aqueous liquid 10 collides withthe diameter-reduced portion 217, and hence shock waves occur. In thisway, due to the existence of the diameter-reduced portion 217, the shockwaves can more frequently occur in the manufacturing container 20,compared to a case where a container having the same inner diameter isused.

Furthermore, in the step of vibrating the manufacturing container 20,the aqueous liquid 10 moves to the bottom body portion 216 from the topbody portion 215, and hence the movement speed of the aqueous liquid 10increases. Accordingly, due to a cavitation effect, the bubbles are moreeasily generated in the aqueous liquid 10 within the bottom body portion216. Due to this synergistic effect, the bubbles 1 having the uniformparticle size can be more efficiently generated in a shorter period oftime.

The screw groove 214 is formed on the entirety of an outercircumferential surface of the bottom body portion 216. Therefore, aslong as the length thereof in the longitudinal direction is within theaforementioned range, the weight portion 5 can be moved to the vicinityof the center from the bottom end of the container body 21.

In a case where the weight portion 5 is provided in the vicinity of thebottom end of the container body 21, the weight of the bottom endportion of the container body 21 increases. Therefore, as in the tenthembodiment described above, in Step (S4), it is possible to increase themagnitude of the shock waves that occur when the aqueous liquid 10collides with the bottom end portion of the container body 21.

In contrast, in a case where the weight portion 5 is positioned in thevicinity of the center of the container body 21, the weight of thediameter-reduced portion 217 increases. Accordingly, in Step (S4), it ispossible to increase the magnitude of the shock waves that occur whenthe aqueous liquid 10 collides with the diameter-reduced portion 217.

The closer the position of the weight portion 5 is to the bottom endportion of the container body 21, the stronger the shock waves thatoccur when the aqueous liquid 10 collides with the bottom end portion ofthe container body 21. That is, in the present embodiment, by adjustingthe position of the weight portion 5 to be provided in the bottom bodyportion 216, the magnitude of the shock waves occurring in themanufacturing container 20 can be controlled, and hence the bubbles 1having an intended size can be stably generated.

The total length of the container body 21 in the longitudinal directionis not particularly limited, but is preferably about 20 to 85 mm andmore preferably about 30 to 53 mm.

The weight portion 5 is a ring-shaped member. By rotating the weightportion 5 in a state where the weight portion 5 is screwed with thescrew groove 214 of the bottom body portion 216, the weight portion 5moves in the region of the bottom body portion 216, in which the screwgroove 214 is formed, in the longitudinal direction (vertical directionin FIG. 17).

As described above, in the method for manufacturing bubbles of thepresent embodiment, by setting the position in which the weight portion5 is provided in the container body 21, the magnitude of the shock wavesoccurring in the manufacturing container 20 can be controlled. In thisway, the size and content of the obtained bubbles 1 can be adjusted.

The mass of the weight portion 5 is not particularly limited, but ispreferably about 3 to 30 g and more preferably about 5 to 20 g for thecontainer body 21 having the aforementioned dimensions. In a case wherethe mass of the weight portion 5 is within the aforementioned range, thesize and content of the obtained bubbles 1 can be more efficientlyadjusted.

The O-rings 6 are provided on the upper side and the lower side of theweight portion 5 as if sandwiching the weight portion 5 therebetween.The O-rings 6 are members that prevent the weight portion 5 from movingdue to the vibration of the manufacturing container 20 or the like. Asthe O-rings 6, O-rings made of silicon can be used.

The weight portion 5 and the O-rings 6 can be mounted on the containerbody 21 in the following manner. First, one of the O-rings 6 is put onthe container body 21 from the bottom end side of the bottom bodyportion 216 and then stopped in a predetermined position. Then, theweight portion 5 is mounted on the bottom end portion of the bottom bodyportion 216 and moved until the weight portion 5 contacts with theO-ring 6 mounted. Then, the other O-ring 6 is put on the container body21 from the bottom end side of the bottom body portion 216 and moveduntil the O-ring 6 contacts with the weight portion 5, and in this way,the weight portion 5 is fixed by the two O-rings 6.

By performing the following steps in the same manner as in the first tofifth embodiments described above, the large amount of bubbles 1 havingthe uniform size can be stably manufactured in the manufacturingcontainer 20. In addition, the manufacturing container (sealedcontainer) 20 (bubble-containing container) containing the large amountof bubbles 1 having the uniform size is obtained.

With the method for manufacturing bubbles of the twelfth embodiment, thesame operations and effects as in the methods for manufacturing bubblesof the first to eleventh embodiments are also obtained.

Thirteenth Embodiment

Next, a thirteenth embodiment of the method for manufacturing bubbles ofthe present invention will be described.

FIG. 18 shows cross-sectional views for illustrating a manufacturingcontainer used in the thirteenth embodiment of the method formanufacturing bubbles of the present invention. FIG. 18(a) shows themanufacturing container in a disassembled state, and FIG. 18(b) showsthe manufacturing container in an assembled state. FIG. 19 shows viewsfor illustrating positions of an opening portion formed in a lid of themanufacturing container shown in FIG. 18(b). FIG. 19(a) is a view forillustrating a state where an injection needle of a syringe is not yetpierced into a rubber stopper, and FIG. 19(b) is a view for illustratinga state where a fastening portion is fastened to a bottom plate portionafter the injection needle is pulled out of the rubber stopper.

In the following description, the upper side in each of FIGS. 18(a) and18(b) will be referred to as “top”, and the lower side in each of FIGS.18(a) and 18(b) will be referred to as “bottom”. Furthermore, the leftside in each of FIGS. 18(a) and 18(b) will be referred to as “left”, andthe right side in each of FIGS. 18(a) and 18(b) will be referred to as“right”.

Hereinafter, regarding the method for manufacturing bubbles of thethirteenth embodiment, the differences between the methods formanufacturing bubbles of the first to twelfth embodiments and the methodfor manufacturing bubbles of the present embodiment will be mainlydescribed, and the same details will not be described.

The method for manufacturing bubbles of the present embodiment is thesame as the methods for manufacturing bubbles of the first to fifthembodiments described above, except that the lid of the manufacturingcontainer has a different constitution.

[S1] Preparation Step

The manufacturing container 20 (the ninth embodiment of the bubblemanufacturing container) shown in FIG. 18(b) is prepared.

The manufacturing container 20 of the present embodiment includes thesame container body 21 as that of the manufacturing container 20 of thefirst embodiment described above, and the lid 22.

In the present embodiment, the lid 22 includes the bottom plate portion223 fixed to the vial mouth of the container body 21, the rubber stopper221 disposed on the bottom plate portion 223 on the side opposite to thecontainer body 21, and the fastening portion 222 fixing the rubberstopper 221 to the bottom plate portion 223.

The bottom plate portion 223 includes a disk-like flat plate portion 225and a cylindrical portion 226 that stands on the edge of the flat plateportion 225. When seen in a cross-sectional view, the bottom plateportion 223 looks like an approximately C-shaped member. In the presentembodiment, when seen in a plan view (top view), the shape of the bottomplate portion 223 (flat plate portion 225) is practically the same asthe shape of the rubber stopper 221, and the diameter of the bottomplate portion 223 is practically the same as that of the rubber stopper221. Furthermore, on the inner circumferential surface of thecylindrical portion 226 and the outer circumferential surface of thecontainer body 21 on the vial mouth side, screw grooves which can bescrewed with each other are formed. By screwing these grooves with eachother, the bottom plate portion 223 (flat plate portion 225) is fixed tothe vial mouth of the container body 21 in a state of adhering to thevial mouth.

When seen in a plan view (top view), in the bottom plate portion 223, athrough hole 224, having a size that allows an injection needle of asyringe to be inserted thereinto, is formed in a position separated fromthe center of the bottom plate portion 223 by a predetermined distance.That is, as shown in FIG. 19(a), when seen in the plan view, in the lid22, the center C of the rubber stopper 221 deviates from the throughhole 224 of the bottom plate portion 223.

Similarly to the bottom plate portion 223 of the lid 22 shown in FIG. 9described above, the aforementioned bottom plate portion 223 isconstituted with a material having a high specific gravity, such as aceramic material and a metal material. By increasing the mass of thebottom plate portion 223, in Step (S4), it is possible to furtherincrease the magnitude of the shock waves that occur when the aqueousliquid 10 collides with the top surface (bottom plate portion 223) ofthe manufacturing container 20. As a result, fine bubbles 1 can be moreeasily and stably generated in the aqueous liquid 10.

As the rubber stopper 221, it is possible to use the same rubber stopperas the rubber stopper 221 used in the method for manufacturing bubblesof the first embodiment described above. On the surface of the rubberstopper 221, in a state where the lid 22 is mounted on the containerbody 21, a mark X is made which is for piercing an injection needle in aposition corresponding to the through hole 224 of the bottom plateportion 223 (see FIG. 19(a)).

The fastening portion 222 is constituted such that it covers the edge ofthe rubber stopper 221. Furthermore, on the outer circumferentialsurface of the fastening portion 222 and the bottom plate portion 223(flat plate portion 225), screw grooves that can be screwed with eachother are formed. By screwing the grooves with each other, the rubberstopper 221 is fixed to the bottom plate portion 223 (flat plate portion225) in a state of adhering to the bottom plate portion 223.

[S3] Step of Sealing Manufacturing Container

First, by using the gas 3, purging is performed in the void portion 11of the container body 21 into which the aqueous liquid 10 is injected.Then, the lid 22 is inserted into the opening portion (vial mouth) ofthe container body 21 (the state shown in FIG. 19(a)). In this way, theaqueous liquid 10 and the gas 3 are sealed in the manufacturingcontainer 20.

In the state shown in FIG. 19(a), an injection needle of a syringefilled with the gas 3 is pierced into the rubber stopper 221 at the markX and inserted into the through hole 224 of the bottom plate portion223. Then, the gas 3 is further added into the manufacturing container20 from the syringe such that the interior of the manufacturingcontainer 20 is pressurized, and the injection needle is pulled out ofthe rubber stopper 221.

Thereafter, by turning the fastening portion 222, the fastening portion222 is fastened to the bottom plate portion 223 (the state shown in FIG.19(b)). By fastening the fastening portion 222 to the bottom plateportion 223, the rubber stopper 221 is compressed toward the bottomplate portion 223 side while rotating (for example, rotating 180°) withrespect to the bottom plate portion 223. Accordingly, when seen in aplan view, a position of a through hole 227 formed in the rubber stopper221 due to the injection needle pierced into the stopper deviates fromthe position of the through hole 224 of the bottom plate portion 223(see FIG. 19(b)). As a result, the through hole 224 of the bottom plateportion 223 is closed due to the rubber stopper 221, and it is possibleto obtain the manufacturing container 20 which is sealed in a statewhere the interior of the manufacturing container 20 is pressurized dueto the gas 3.

In the present embodiment, there is no portion that makes the interiorof the manufacturing container 20 communicate with the outside, andhence the sealing property of the interior of the manufacturingcontainer 20 can be improved. Due to the improvement of the sealingproperty of the interior of the manufacturing container 20, the bubbles1 can more stably exist in the aqueous liquid 10 within the finallyobtained bubble-containing container. That is, the long-term storabilityof the bubble-containing container is further improved.

As described above, by fastening the fastening portion 222 to the bottomplate portion 223, the rubber stopper 221 is compressed toward thebottom plate portion 223 side. Provided that a thickness of the rubberstopper 221 in a state where the fastening portion 222 is not yetfastened to the bottom plate portion 223 is t₁ (mm), and that athickness of the rubber stopper 221 in a state where the fasteningportion 222 is fastened to the bottom plate portion 223 is t₂ (mm), acompression rate ((t₁−t₂)/t₁×100) of the rubber stopper 221 ispreferably 5% to 60%, and more preferably 10% to 30%. In a case wherethe compression rate is as described above, it is possible to furtherimprove the adhesion between the rubber stopper 221 and the bottom plateportion 223 while suppressing the load imposed on the rubber stopper 221due to the fastening of the fastening portion 222. As a result, thesealing property of the interior of the manufacturing container 20 canbe further improved.

In the present embodiment, the large amount of bubbles 1 having theuniform size can also be stably manufactured in the manufacturingcontainer 20. In addition, the manufacturing container 20(bubble-containing container) containing the large amount of bubbles 1having the uniform size is obtained. A plurality of through holes 224may be provided in the bottom plate portion 223 in the same manner asthe sixth embodiment described above.

With the method for manufacturing bubbles of the thirteenth embodiment,the same operations and effects as in the methods for manufacturingbubbles of the first to twelfth embodiments are also obtained.

Hitherto, the method for manufacturing bubbles and the bubblemanufacturing container of the present invention have been describedbased on the embodiments illustrated in drawings. However, the presentinvention is not limited thereto, and each step can be substituted withany step that can perform the same function.

For example, certain constitutions of the first to thirteenthembodiments can be combined with each other.

EXAMPLES

In order to explain the influence of the volume of the gas 3 sealed inthe manufacturing container 20 and the number of revolution of themanufacturing container 20 on the diameter and content of the bubble 1generated in the aqueous liquid 10, the following experiments wereperformed.

Example 1

First, relationships between the number of revolution of themanufacturing container 20 and the diameter and content of the bubbles 1generated in the aqueous liquid 10 were investigated.

(Method for Manufacturing Bubbles)

[Preparation Step]

First, 120 μl of an albumin solution (ALBUMINAR 25% manufactured by CSLBehring) containing albumin at 250 mg/ml and 12 ml of 25%phosphate-buffered saline (PBS) were prepared. Furthermore, a 15 ml vial(height X: 50 mm, outer diameter R: 25 mm) was prepared. The vial hadthe same shape as that of the manufacturing container 20 shown in FIG.4.

[Step of Injecting Aqueous Liquid into Container]

The entireties of the albumin solution and the 25% phosphate-bufferedsaline were injected into the prepared vial. Here, a height Y of asurface of an aqueous liquid obtained by mixing the albumin solutionwith the 25% phosphate-buffered saline was 25 mm.

[Step of Sealing Container]

Then, by using perfluorobutane, purging was performed in the void in thevial into which the aqueous liquid was injected, and then a lid havingthe same shape as that of the lid 22 shown in FIG. 4 was inserted intothe mouth of the vial. Thereafter, a syringe filled with perfluorobutanewas prepared. An injection needle of the syringe was pierced into therubber stopper of the lid, and 2 ml of perfluorobutane was further addedinto the vial from the syringe. In this way, a sealed vial having aninternal pressure of 2 atm was obtained.

[Step of Vibrating Container]

Then, two sealed vials obtained as above were prepared. By usingPrecellys (high-speed cell disruption system) manufactured by bertinTechnologies, one of the sealed vials was vibrated for 30 seconds at thenumber of revolution of 5,000 rpm, and the other vial was vibrated for30 seconds at the number of revolution of 6,500 rpm. At this time, thesealed vials were caused to reciprocate in a vertical direction, and itwas confirmed that the aqueous liquid repeatedly collided with the topand bottom surfaces of the vial. When the sealed vials were vibrated, avibration width of the sealed vials was 40 mm in the longitudinaldirection (vertical direction) and 20 mm in the transverse direction(horizontal direction). The conditions were set as described above suchthat an instantaneous relative speed between each vial and the aqueousliquid became equal to or higher than 40 km/h in any of the vials.

[Step of Allowing Container to Stand]

After being vibrated, the sealed vials were allowed to stand, therebyobtaining bubble-containing containers.

(Measurement of Bubble Diameter Distribution)

From each of the bubble-containing containers obtained as above, theaqueous liquid containing bubbles (bubble-containing liquid) was takenusing a syringe. Then, by using a bubble measurement device(nanoparticle analysis system nanosight), a bubble diameter distributionof the bubbles contained in the aqueous liquid was measured. The resultsare shown in FIG. 20.

FIG. 20(a) is a graph showing a bubble diameter distribution of bubblesmanufactured at the number of revolution of each of 5,000 rpm and 6,500rpm. FIG. 20(b) is a partially enlarged view obtained by setting therange of the abscissa axis in the graph shown in FIG. 20(a) to be 0 to700 nm.

As shown in FIG. 20(a), in a case where the sealed vial was vibrated at6,500 rpm, the content of the bubbles in the aqueous liquid can befurther increased greatly than in a case where the sealed vial wasvibrated at 5,000 rpm. Particularly, the content of bubbles having asmaller diameter than about 600 nm was greater in a case where thesealed vial was vibrated at 6,500 rpm than in a case where the sealedvial was vibrated at 5,000 rpm, in more than 3 to 5 times.

In a case where the number of revolution of the sealed vial was 5,000rpm, by lengthening the vibration time, the content of the bubbles inthe aqueous liquid can be increased to some extent. However, the contentof the bubbles obtained in this aqueous liquid was smaller than thecontent of the bubbles obtained in a case where the number of revolutionwas 6,500 rpm.

As shown in FIG. 20(b), in a case where the sealed vial was vibrated at6,500 rpm, a large amount of extremely small bubbles having a diameterof about 100 to 150 nm could be generated.

The above results are considered to be yielded by the followingoperations and effects. That is, according to the number of revolutionof the sealed vial, the magnitude of the pressure of the shock wavesthat occur when the aqueous liquid collides with the vial changes. Themagnitude of the pressure of the shock waves is an important factor thatdetermines the diameter and the content of the bubbles generated in theaqueous liquid. In a case where the vial is stirred using a generalstirrer or vibrated at the number of revolution lower than 5,000 rpm,such shock waves do not occur, or even though the shock waves occur, theamount of the occurring shock waves is small. Therefore, unlike in theinvention of the present application, the bubbles having a sufficientlysmall diameter cannot be generated in the aqueous liquid at a highcontent.

FIG. 21 shows results obtained by analyzing a graph of a bubble diameterdistribution shown in FIG. 20(a). FIG. 21(a) is a graph showing arelationship between the number of revolution of a sealed vial and anaverage bubble diameter. FIG. 21(b) is a graph showing the relationshipbetween the number of revolution of a sealed vial and a content ofbubbles.

As shown in FIG. 21(a), in a case where the sealed vial was vibrated at6,500 rpm, an average diameter of the generated bubbles was smaller thanin a case where the sealed vial was vibrated at 5,000 rpm, by about 80nm. Furthermore, as shown in FIG. 21(b), in a case where the sealed vialwas vibrated at 6,500 rpm, a content of the generated bubbles wassmaller than in a case where the sealed vial was vibrated at 5,000 rpm,by about 9×10⁷ (particle)/ml. From these results, it was also understoodthat in a case where the sealed vial is vibrated at 6,500 rpm, morebubbles having a small diameter could be generated as compared with acase where the sealed vial is vibrated at 5,000 rpm.

Example 2

Then, relationships between the volume of the gas 3 sealed in themanufacturing container 20 and the diameter and content of the bubbles 1generated in the aqueous liquid 10 were investigated.

(Method for Manufacturing Bubbles)

A bubble-containing container was obtained in the same manner as inExample 1, except that in Step of sealing container in Example 1, foursealed vials were prepared, which were obtained by changing the volumeof perfluorobutane sealed in the vial into 0.5 ml, 1 ml, 1.5 ml, and 2ml, respectively.

Regarding each of the sealed vials, the following Table 1 shows thevolume (ml) of the gas (perfluorobutane) sealed therein, the internalpressure (atm) of the sealed vial, and the number of revolution (rpm) ofthe sealed vial in Step of vibrating container.

TABLE 1 Volume of gas Internal pressure Number of to be sealed of sealedvial revolution (ml) (atm) (rpm) 0.5 1.2 6,500 1 1.3 6,500 1.5 1.5 6,5002 2 6,500

(Measurement of Bubble Diameter Distribution)

In the same manner as in Example 1, a bubble diameter distribution ofthe bubble-containing liquid in each of the obtained bubble-containingcontainers was measured. FIG. 22 shows results obtained by analyzing agraph of the obtained bubble diameter distribution.

FIG. 22(a) is a graph showing a relationship between a volume of a gassealed in a sealed vial and an average diameter of bubbles.

FIG. 22(b) is a graph showing a relationship between a volume of a gassealed in a sealed vial and a content of bubbles.

As shown in FIG. 22(a), even though the number of revolution at whicheach of the sealed vials was vibrated was the same, by increasing theinternal pressure of the sealed vial, the average diameter of thegenerated bubbles was reduced. Specifically, in a case where theinternal pressure of a sealed vial was 2 atm, the average diameter ofthe generated bubbles was smaller than in a case where the internalpressure of the sealed vial was 1.2 atm, by about 100 nm.

As shown in FIG. 22(b), by increasing the internal pressure of thesealed vial, the content of the bubbles increased. Particularly, in acase where the internal pressure of a sealed vial was 2 atm, the contentof the bubbles was greater than in a case where the internal pressure ofthe sealed vial was 1.2 atm, in equal to or more than 2.

It is considered that in a case where the internal pressure of thesealed vial is increased, the pressure of the shock waves which occurwhen the aqueous liquid collides with the vial may exert a big influenceon the diameter and content of the generated bubbles. Therefore, eventhough the number of revolution at which each of the sealed vials isvibrated is the same, according to the internal pressure of the sealedvial, the average diameter of the generated bubbles changes.Furthermore, in a case where the internal pressure of the sealed vialincreases, a large amount of gas is incorporated into the aqueousliquid. Consequently, due to the influence of the shock waves describedabove, the content of the bubbles generated in the aqueous liquid couldbe increased.

Example 3

(Method for Manufacturing Nanobubbles Containing GFP Gene)

[Preparation Step]

First, in the same manner as in the first embodiment described above,120 μl of an albumin solution (ALBUMINAR 25% manufactured by CSLBehring) and 12 ml of 25% phosphate-buffered saline were prepared.Furthermore, 2 μg of GFP genes were prepared. In addition, a 15 ml vial(height X: 50 mm, outer diameter R: 25 mm) was prepared. The vial hadthe same shape as that of the manufacturing container 20 shown in FIG.4.

[Step of Injecting Aqueous Liquid into Container]

The entireties of the albumin solution, the 25% phosphate-bufferedsaline, and the GFP genes were injected into the prepared vial. Here, aheight Y of a surface of an aqueous liquid obtained by mixing togetherthe albumin solution, the 25% phosphate-buffered saline, and the GFPgenes was 25 mm.

[Step of Sealing Container]

Then, by using perfluorobutane, purging was performed in a void in thevial into which the aqueous liquid was injected, and then a lid havingthe same shape as that of the lid 22 shown in FIG. 4 was inserted intothe mouth of the vial. Thereafter, a syringe filled with perfluorobutanewas prepared. An injection needle of the syringe was pierced into therubber stopper of the lid, and 2 ml of perfluorobutane was further addedinto the vial from the syringe. In this way, a sealed vial having aninternal pressure of 2 atm was obtained.

[Step of Vibrating Container]

Then, by using Precellys manufactured by bertin Technologies, the sealedvial was vibrated for 30 seconds at the number of revolution of 7,000rpm. At this time, the sealed vial was caused to reciprocate in avertical direction, and it was confirmed that the aqueous liquidrepeatedly collided with the top and bottom surfaces of the vial. Whenthe sealed vial was vibrated, a vibration width of the sealed vial was40 mm in the longitudinal direction (vertical direction) and 20 mm inthe transverse direction (horizontal direction). The conditions were setas described above such that an instantaneous relative speed between thevial and the aqueous liquid became equal to or higher than 40 km/h.

[Step of Allowing Container to Stand]

After being vibrated, the sealed vial was allowed to stand, therebyobtaining a bubble-containing container. The aqueous liquid(bubble-containing liquid) containing bubbles was taken out using asyringe, and by using a bubble measurement device (nanoparticle analysissystem nanosight), the sizes of the bubbles were confirmed. As a result,an average diameter of the bubbles was 600 nm.

<Evaluation of Introduction of Fluorescent Protein Expression Gene intoCell>

0.2 μg of the aqueous liquid obtained in Example 3 was added to a petridish in which cerebrovascular pericytes (manufactured by TAKARA BIO INC,product code: C-12980) were cultured, thereby obtaining a culture mediumof the cerebrovascular pericytes. Pericytes are known as cells intowhich a gene is hardly introduced.

Four samples of the culture medium of the cerebrovascular pericytes wereprepared. These samples were irradiated with ultrasonic waves having afrequency of 1.0 MHz (sine waves, pulse repetition frequency (PRF): 100Hz, duty cycle (DC): 10%) for 60 seconds at the following power ofirradiation.

[Power of Irradiation]

0.6 W/cm², 0.8 W/cm², 0.9 W/cm², 1.0 W/cm²

Thereafter, the culture mediums of the cerebrovascular pericytes werecultured for 48 hours at 37° C., and then each of the samples obtainedin this way was observed with a fluorescence microscope.

FIG. 23 shows fluorescent micrographs of a culture medium ofcerebrovascular pericytes cultured for 48 hours at 37° C. FIG. 23(a) isan image of a sample irradiated with ultrasonic waves at an irradianceof 0.6 W/cm², and FIG. 23(b) is an image of a sample irradiated withultrasonic waves at an irradiance of 0.8 W/cm². Furthermore, FIG. 24shows fluorescent micrographs of a culture medium of cerebrovascularpericytes cultured for 48 hours at 37° C. FIG. 24(a) is an image of asample irradiated with ultrasonic waves at an irradiance of 0.9 W/cm²,and FIG. 24(b) is an image of a sample irradiated with ultrasonic wavesat an irradiance of 1.0 W/cm².

As shown in FIGS. 23(a) and 23(b) and FIGS. 24(a) and 24(b), in any ofthe samples irradiated with ultrasonic waves at any power ofirradiation, a region developing green was confirmed. This shows that agreen fluorescent protein (GFP) is expressed in the cerebrovascularpericytes in each sample. Accordingly, it was understood that in any ofthe sample, the bubbles burst due to the irradiation of the ultrasonicwaves, and hence the GFP genes contained in the bubbles wereincorporated into the cerebrovascular pericytes.

Then, relationships between the type of the aqueous liquid 10 and thediameter and content of the bubbles 1 generated in the aqueous liquid 10were investigated.

Example 4

(Method for Manufacturing Bubbles)

[Preparation Step]

First, as an aqueous liquid, 12 ml of distilled water was prepared.Furthermore, a 15 ml vial (height X: 50 mm, outer diameter R: 25 mm) wasprepared. The vial has the same shape as that of the manufacturingcontainer 20 shown in FIG. 4.

[Step of Injecting Aqueous Liquid into Container]

The distilled water (aqueous liquid) was injected into the preparedvial. A height Y of a surface of the aqueous liquid was 25 mm.

Then, by using perfluoropropane as a gas to be filled into the vial,[Step of sealing container] was performed. Thereafter, in the samemanner as in the first embodiment, [Step of vibrating container] and[Step of allowing container to stand] were performed, thereby obtaininga bubble-containing container.

Example 5

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to a 1 w/v %aqueous dextran solution.

Example 6

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to 100%phosphate-buffered saline (PBS).

Example 7

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to SOLDEM 3Ainfusion solution (manufactured by Terumo Corporation).

Example 8

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to SOLDEM 1infusion solution (manufactured by Terumo Corporation).

Example 9

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to physiologicalsaline (0.9 w/v % aqueous NaCl solution).

Example 10

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to a 0.25 w/v %aqueous albumin solution.

Example 11

A bubble-containing container was obtained in the same manner as inExample 4, except that the distilled water was changed to a 20 w/v %aqueous glucose solution.

(Measurement of Bubble Diameter Distribution)

For each of the bubble-containing liquids of Examples 4 to 11 obtainedas above, the bubble diameter distribution was measured in the samemanner as in Example 1. FIG. 25 shows the results.

FIG. 25 is a graph showing bubble diameter distributions of the bubblesobtained in Examples 4 to 11.

As shown in FIG. 25, it was understood that the higher the concentrationof water in the aqueous liquid is, the smaller the diameter of thegenerated bubbles 1 tends to be, and the larger the amount of thebubbles generated tends to be. Particularly, in Example 4 in whichdistilled water was used as the aqueous liquid, the average diameter ofthe bubbles was about 100 nm, and the content of the bubbles generatedin the bubble-containing liquid was about 27×10⁶ particles/ml. Morespecifically, in Example 4, the bubbles having a bubble diameter ofabout 100 nm were generated the most. Furthermore, in Example 4, as apeak of the diameter of 100 nm, the bubbles having the bubble diameterof about 0 to 200 nm were generated.

In Example 7 in which the SOLDEM 3A infusion solution was used as theaqueous liquid and in Example 8 in which the SOLDEM 1 infusion solutionwas used as the aqueous liquid, the number of generated bubbles wassmaller than in Example 4. However, in each of Examples 7 and 8, thebubble diameter distribution having a shape similar to that of Example 4was obtained.

In Example 11 in which the 20 w/v % aqueous glucose solution was used asthe aqueous liquid, the bubbles having the bubble diameter of about 200nm were generated the most. The bubbles obtained in Example 11 had abroad bubble diameter distribution and the bubble diameter of about 100to 400 nm. In Example 10 in which the 0.25% albumin was used as theaqueous liquid, the bubbles having the bubble diameter of about 100 to500 nm were evenly generated.

In all of the bubble-containing liquids of Examples 4 to 11, bubbleshaving a size of equal to or larger than 500 nm substantially did notexist. Therefore, by using such bubble-containing liquids as anultrasound contrast agent, a high-definition image with high resolutioncan be obtained.

Then, relationships between the type of the gas 3 and the diameter andcontent of the bubbles 1 generated in the aqueous liquid 10 wereinvestigated.

Example 12

In Example 4, by changing the distilled water to physiological saline(0.9 w/v % aqueous NaCl solution), [Preparation step] and [Step ofinjecting aqueous liquid into container] were performed. Then, by usingair as a gas to be filled into the vial, [Step of sealing container] wasperformed. Thereafter, in the same manner as in the first embodiment,[Step of vibrating container] and [Step of allowing container to stand]were performed, thereby obtaining a bubble-containing container.

Example 13

A bubble-containing container was obtained in the same manner as inExample 12, except that the air was changed to ethylene (C₂H₄).

Example 14

A bubble-containing container was obtained in the same manner as inExample 13, except that hydrogen was changed to ethylene (C₂H₄).

Example 15

A bubble-containing container was obtained in the same manner as inExample 13, except that hydrogen was changed to ethane (C₂H₆).

Example 16

A bubble-containing container was obtained in the same manner as inExample 13, except that hydrogen was changed to methane (CH₄).

Example 17

A bubble-containing container was obtained in the same manner as inExample 13, except that hydrogen was changed to nitrous oxide (N₂O).

(Measurement of Bubble Diameter Distribution)

For each of the bubble-containing liquids of Examples 12 to 17 obtainedas above, a bubble diameter distribution was measured in the same manneras in Example 1. FIG. 26 shows the results.

FIG. 26 is a graph showing bubble diameter distributions of bubblesobtained in Examples 12 to 17.

As shown in FIG. 26, by changing the type of the gas sealed in the vial,the amount of bubbles generated changed. In a case (Example 12) wherethe gas sealed in the vial was ethylene, the amount of bubbles generatedwas larger than in a case (Example 14) where the air was used as the gassealed in the vial, by about 2 times. Particularly, it was understoodthat in Examples 13 and 15 in which hydrogen and ethane were used as thegas, respectively, the amount of generated bubbles increased.

Then, the diameter and content of the bubbles 1 generated using themanufacturing container 20 shown in FIG. 17 were investigated.

Example 18

(Method for Manufacturing Bubbles)

[Preparation Step]

First, in the same manner as in the first embodiment described above,120 μl of an albumin solution (ALBUMINAR 25% manufactured by CSLBehring) and 12 ml of 25% phosphate-buffered saline were prepared.Furthermore, the manufacturing container 20 (length of top body portion:23.82 mm, inner diameter of top body portion: 10.26 mm, length of bottombody portion: 20 mm, inner diameter of bottom body portion: 5.9 mm)shown in FIG. 17 was prepared.

[Step of Injecting Aqueous Liquid into Container]

The entireties of the albumin solution and the 25% phosphate-bufferedsaline were injected into the prepared manufacturing container. A heightof a surface of an aqueous liquid obtained by mixing the albuminsolution with the 25% phosphate-buffered saline was 25 mm from thebottom surface of the manufacturing container 20.

[Step of Sealing Container]

Then, a lid having the same shape as that of the lid 22 shown in FIG. 17was inserted into the vial mouth (opening portion) of the manufacturingcontainer 20 into which the aqueous liquid was injected. In this way, asealed container (manufacturing container 20 sealed) having an internalpressure of 1 atm was obtained.

[Step of Vibrating Container]

Thereafter, two sealed containers described above were prepared. Theweight portion 5 weighing 13.5 g was mounted on only one of the sealedcontainers (manufacturing containers 20). By using Precellysmanufactured by bertin Technologies, these two manufacturing containerswere vibrated for 30 seconds at the number of revolution of 6,500 rpm.At this time, the sealed containers were caused to reciprocate in avertical direction, and it was confirmed that the aqueous liquidrepeatedly collides with the top and bottom surfaces of the sealedcontainers. When the sealed containers were vibrated, a vibration widthof the sealed containers was 40 mm in the longitudinal direction(vertical direction) and 20 mm in the transverse direction (horizontaldirection). The conditions were set as described above such that aninstantaneous relative speed between manufacturing container 20 and theaqueous liquid became equal to or higher than 40 km/h.

[Step of Allowing Container to Stand]

After being vibrated, the sealed containers were allowed to stand,thereby obtaining bubble-containing containers.

Example 19

In the same manner as in Example 18, an aqueous liquid formed of analbumin solution and 25% phosphate-buffered saline was injected into themanufacturing container 20 shown in FIG. 17.

Then, by filling the manufacturing container 20 with air in the samemanner as in Example 12, [Step of sealing container] was performed.Thereafter, in the same manner as in Example 18, [Step of vibratingcontainer] and [Step of allowing container to stand] were performed,thereby obtaining a bubble-containing container.

Example 20

A bubble-containing container was obtained in the same manner as inExample 19, except that the air was changed to perfluoropropane.

(Measurement of Bubble Diameter Distribution)

For each of the bubble-containing liquids of Examples 18 to 20 obtainedas above, a bubble diameter distribution was measured in the same manneras in the first embodiment. Furthermore, a few drops of each of thebubble-containing liquids obtained in Examples 18 to 20 were added to aprepared slide by using a syringe, and it was observed with an opticalmicroscope. FIGS. 27 and 28 show the results.

FIG. 27(a-1) shows a micrograph of the bubbles obtained using acontainer without the weight in Example 18 and a graph showing thebubble diameter distribution. FIG. 27(a-2) shows a micrograph of thebubbles obtained using the container with the weight in Example 18 and agraph showing the bubble diameter distribution. FIG. 27(b-1) shows amicrograph of the bubbles obtained using a container without the weightin Example 19 and a graph of the bubble diameter distribution. FIG.27(b-2) shows a micrograph of the bubbles obtained using the containerwith the weight in Example 19 and a graph of the bubble diameterdistribution.

FIG. 28(a-1) shows a micrograph of the bubbles obtained using acontainer without the weight in Example 20 and a graph of the bubblediameter distribution. FIG. 28(a-2) shows a micrograph of the bubblesobtained using the container with the weight in Example 20 and a graphof the bubble diameter distribution.

In the graphs of the bubble diameter distributions shown in each ofFIGS. 27 and 28, the abscissa axis shows the measured bubble diameter.The diameter increases toward the right side from the left side on theabscissa axis. In the graph, the leftmost bar on the abscissa axis showsthe amount of bubbles having an average diameter of equal to or lessthan 1 μm.

As shown in FIGS. 27 and 28, in all of Examples 18 to 20, in a casewhere the container with the weight was used, the amount of generatedbubbles increased. Particularly, it was understood that bubbles having asmall diameter (diameter: equal to or less than 1 μm) were generated ina markedly large amount. Furthermore, from the comparison between FIG.27(b-2) and FIG. 28(c-2), it was understood that in the case whereperfluoropropane was used as the gas sealed in the container, thebubbles having a smaller diameter were generated more than in a casewhere air was used as a gas. Accordingly, it was understood that byusing the manufacturing container with the weight mounted on a portionthereof as shown in FIG. 17, the bubbles having the small diameter(bubbles having a diameter of equal to or less than 1 μm) could beefficiently manufactured.

INDUSTRIAL APPLICABILITY

According to the bubble manufacturing container of the presentinvention, simply by vibrating the container at a predetermined numberof revolution, a large amount of bubbles having a uniform size can bestably generated in an aqueous liquid. The bubble obtained in this waycan be used in various fields such as medical care, food, seafoodfarming, and waste water treatment. Accordingly, the bubblemanufacturing container of the present invention is industriallyapplicable.

1. A bubble manufacturing container used for manufacturing bubbles,comprising: a container body having an opening portion; and a rubberstopper provided on the opening portion of the container body, whereinthe rubber stopper is constituted so that the bubbles of an inside ofthe container body are able to be taken by piercing an injection needle.2. The bubble manufacturing container according to claim 1 furthercomprising a fastening portion provided on the rubber stopper, having anopening and sealing the container body with the rubber stopper, whereinthe container body mounts a weight portion.
 3. The bubble manufacturingcontainer according to claim 2, wherein the container body isconstituted from a top body portion having the opening portion and abottom body portion mounting the weight portion, and wherein the bottombody portion has an inner diameter smaller than an inner diameter of thetop body portion.
 4. The bubble manufacturing container according toclaim 3, wherein the top body portion has a diameter-reduced portionthat the inner diameter of the top body portion is reduced so as tobecome the inner diameter of the bottom body portion.
 5. The bubblemanufacturing container according to claim 3, wherein the bottom bodyportion has a screw groove formed over an entirety of an outercircumferential surface of the bottom body portion, and wherein theweight portion is constituted to screw with the screw groove and bemovable on the bottom body portion.
 6. The bubble manufacturingcontainer according to claim 2, wherein the weight portion is providedin a vicinity of the opening portion of the container body and has athrough hole to be pierced by the injection needle, and the through holecorresponds to the opening of the fastening portion.
 7. The bubblemanufacturing container according to claim 6, wherein the weight portionis provided on the container body to cover the opening portion, andwherein the rubber stopper is provided on the weight portion and has amark to be pierced by the injection needle at a position to correspondto the through hole.
 8. The bubble manufacturing container according toclaim 7, wherein the position of the mark of the rubber stopper isconstituted to be shifted from a position of the through hole of theweight portion by rotating the fastening portion.
 9. The bubblemanufacturing container according to claim 1, wherein the container bodyhas a long shape, both end portions and a projection portion formedbetween the both end portions, and the container body has two weightportions covering the both end portions of the container body, andwherein the opening portion is provided on the projection portion. 10.The bubble manufacturing container according to claim 9, wherein thecontainer body is formed into a cylindrical shape to open the both endportions to an outside.
 11. The bubble manufacturing container accordingof claim 2, wherein the weight portion is constituted of a materialhaving a density higher than a density of a material constituting thecontainer body.
 12. The bubble manufacturing container according toclaim 1 further comprising: a mininert valve for maintaining a sealingproperty of the inside of the container body; and a tube forcommunicating the inside of the container body with the mininert valve.13. The bubble manufacturing container according to claim 12, whereinthe mininert valve has a duct communicating with the tube and allowingthe injection needle to be pierced and an opening and closing mechanismcontrolling an opening and closing of the duct, and wherein the tubeconnects to the rubber stopper or the container body.
 14. The bubblemanufacturing container according to claim 1, wherein the container bodyhas an inner surface, and at least a part of the inner surface is in aform of a concave surface, a convex surface or a corrugated surface.